CA2535515C - Cd20-binding polypeptide compositions - Google Patents

Cd20-binding polypeptide compositions Download PDF

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CA2535515C
CA2535515C CA2535515A CA2535515A CA2535515C CA 2535515 C CA2535515 C CA 2535515C CA 2535515 A CA2535515 A CA 2535515A CA 2535515 A CA2535515 A CA 2535515A CA 2535515 C CA2535515 C CA 2535515C
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amino acid
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Francis J. Carr
Stephen Williams
Stephen D. Gillies
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Merck Patent GmbH
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    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/02Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • AHUMAN NECESSITIES
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    • A61P29/00Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • A61P35/02Antineoplastic agents specific for leukemia
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
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    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07K2317/20Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/00Immunoglobulins specific features
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    • C07K2317/52Constant or Fc region; Isotype
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    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732Antibody-dependent cellular cytotoxicity [ADCC]
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    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734Complement-dependent cytotoxicity [CDC]
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S424/00Drug, bio-affecting and body treating compositions
    • Y10S424/80Antibody or fragment thereof whose amino acid sequence is disclosed in whole or in part
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S424/00Drug, bio-affecting and body treating compositions
    • Y10S424/801Drug, bio-affecting and body treating compositions involving antibody or fragment thereof produced by recombinant dna technology

Abstract

The invention relates to polypeptide compositions that bind to CD20 antigen, especially human CD20 antigen. The polypeptide composistions have the biological activity of known anti-CD20 antibodies, such as 2B8 or Leu16, but show a reduced immunogenicity as compared with the non-~modified molecules.
The polypeptide compositions include chimeric antibodies, antibody fragments, and fusion proteins of an antibody or antibody fragment with a cytokine.

Description

, FIELD OF THE INVENTION
The present invention relates to polypeptide compositions that bind to CD20 antigen.
More specifically, the invention relates to polypeptide compositions that bind to human CD20 antigen and methods for using the compositions. Such polypeptide compositions include chimeric antibodies, antibody fragments, and fusion proteins of an antibody or antibody fragment with a cytokine.
BACKGROUND OF THE INVENTION
There are many instances where the efficacy of a therapeutic protein is limited by an unwanted immune reaction to the therapeutic protein. Several mouse monoclonal antibodies have shown promise as therapies in a number of human disease settings but in certain cases have failed due to the induction of significant degrees of a human anti-murine antibody (HAMA) response (Schroff, R. W. et al. (1985) Cancer Res. 45:

885; Shawler, D.L. et al. (1985) J. Immunol. 135: 1530-1535). For monoclonal antibodies, a number of techniques have been developed in attempt to reduce the HAMA response (W0A8909622; EPA0239400; EPA0438310; W0A9106667;
EPA0699755). These recombinant DNA approaches have generally reduced the mouse genetic information in the final antibody construct whilst increasing the human genetic information in the final construct to result in antibody molecules which are generally termed "humanised" antibodies.
Humanised antibodies, for the most part, are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or primate, and this process is sometimes termed "CDR
grafting".
Generally additional Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues and in some instances other substitutions are made to further restore the antibody function.
Typically humanised antibodies are reconstituted into whole antibody molecules comprising two variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanised =
CONFIRMATION COPY
antibody will also generally comprise at least a portion of a human derived immunoglobulin constant region (Pc) (Jones et al. (1986), Nature 321: 522-525;

Reichmann et al. (1988), Nature 332: 323-329). Notwithstanding, humanised antibodies have, in several cases, still elicited an immune response in patients (Issacs J.D. (1990) Sem. Inununol. 2: 449, 456; Rebello, P.R. et al. (1999) Transplantation 68:
1417-1420).
Key to the induction of an immune response is the presence within the protein of peptides that can stimulate the activity of T-cells via presentation on MHC
class II
molecules, so-called "T-cell epitopes". Such T-cell epitopes are commonly defined as any amino acid residue sequence with the ability to bind to MHC Class II
molecules.
Implicitly, a "T-cell epitope" means an epitope which when bound to MHC
molecules can be recognized by a T-cell receptor (TCR), and which can, at least in principle, cause the activation of these T-cells by engaging a TCR to promote a T-cell response.
MHC Class II molecules are a group of highly polymorphic proteins which play a central role in helper T-cell selection and activation. The human leukocyte antigen group DR (HLA-DR) are the predominant isotype of this group of proteins and isotypes HLA-DQ and HLA-DP perform similar functions. In the human population, individuals bear two to four DR alleles, two DQ and two DP alleles. The structure of a number of DR molecules has been solved and these appear as an open-ended peptide binding groove with a number of hydrophobic pockets which engage hydrophobic residues (pocket residues) of the peptide (Brown et al. Nature (1993) 364: 33;
Stern et al. (1994) Nature 368: 215). Polymorphism identifying the different allotypes of class II molecule contributes to a wide diversity of different binding surfaces for peptides within the peptide binding grove and at the population level ensures maximal flexibility with regard to the ability to recognize foreign proteins and mount an immune response to pathogenic organisms.
An immune response to a therapeutic protein proceeds via the MHC class II
peptide presentation pathway. Here exogenous proteins are engulfed and processed for presentation in association with MHC class II molecules of the DR, DQ or DP
type.
MHC Class II molecules are expressed by professional antigen presenting cells (APCs), such as macrophages and dendritic cells amongst others. Engagement of a MHC= class U peptide complex by a cognate T-cell receptor on the surface of the T-cell, together with the cross-binding of certain other co-receptors such as the CD4 molecule, can induce an activated state within the T-cell. Activation leads to the release of cytoldnes further activating other lymphocytes such as B cells to produce antibodies or activating T killer cells as a full cellular immune response.
=
T-cell epitope identification is the first step to epitope elimination.
Identified epitopes may be eliminated by judicious amino acid substitution or other modification strategies. Such an approach is recognized in W098/52976 and W000/34317 where in the latter case computational threading techniques are described as a means to identify polypeptide sequences with the potential to bind a sub-set of human MHC
class II DR allotypes and the predicted T-cell epitopes are removed by amino acid substitution within the protein of interest.
It would be desirable to identify and to remove, or at least to reduce, T-cell epitopes from a given, in principle therapeutically valuable, but originally immunogenic peptide, polyp eptide or protein. One of these therapeutically valuable molecules is a monoclonal antibody with binding specificity for the human B-cell antigen CD20. The preferred monoclonal antibodies of the present invention are modified forms of the antibody 2B8 and Leu16. Antibody 2B8 is described in U.S patent No. 5,736,137, Antibody Leu16 is described in Wu et al. Protein Engineering (2001) 14:1025-1033.
CD20 is a non- glycosylated phosphoprotein of 35,000 Daltons, typically designated as the human B lymphocyte restricted differentiation antigen Bp35B. The protein is a highly cell specific surface molecule expressed on pre-B and mature B-cells including greater than 90% of B-cell non-Hodgkin's lymphomas (NHL). Monoclonal antibodies and radioimmunoconjugates targeting CD20 have emerged as new treatments for NHL. The most significant example includes the parental antibody of the present invention namely monoclonal antibody 2B8 (Reff, M.E. et al. (1994) Blood 83:

445). The variable region domains of 2B8 have been cloned and combined with human constant region domains to produce a chimeric antibody designated C2B8 which is marketed as RITUXANTm in the USA or MABTHERA (rituximab) in Europe. C2B8 is recognized as a valuable therapeutic agent for the treatment of NHL
and other B-cell diseases (Maloney, D.G. et al. (1997) J. Clin. Oncol. 15:
3266-3274;
Maloney, D.G. et al. (1997) Blood 90: 2188-2195).
An additional example of an anti-CD20 therapeutic is provided by the antibody B1, described in US patent No. 6,090,365. This antibody has similarly achieved registration for use as a NHL therapeutic although in this case the molecule (BEXXARTM) is a 1311 radioimmunoconjugate. The native B1 (non-conjugated) antibody has utility in ex vivo purging regimens for autologous bone marrow transplantation therapies for lymphoma and refractory leukaemia (Freedman, A.S. et al. (1990), 1 Clin. Oncol. 8: 784).
Despite the success of antibodies such as C2B8 (rituximab) and BEXXARTm there is a continued need for anti-CD20 analogues with enhanced properties. There is a particular need for enhancement of the in vivo characteristics when administered to the human subject. In this regard, it is highly desired to provide anti-CD20 antibodies with reduced or absent potential to induce an immune response in the human subject.
Such proteins would display an increased circulation time within the human subject and would be of particular benefit in chronic use settings such as is the case for the therapeutic use of anti-CD20 molecules. The present invention provides modified anti-CD20 antibodies that display enhanced properties in vivo.
SUMMARY OF THE INVENTION
The present invention provides polypeptide compositions that bind to CD20 antigen, preferably human CD20 antigen. The CD20 compositions comprise one or more anti-CD20 heavy chain and/or light chain variable region polypeptide segments, which can be polypeptide selected from the group consisting of a modified form of the heavy chain variable region (Vh) of anti-CD20 antibody 2B8, a modified form of the light chain variable region (Vk) of anti-CD20 antibody 2B8, a modified form of the heavy chain variable region (Vh) of anti-CD20 antibody Leul6 and a modified form of the light chain variable region (Vk) of anti- CD20 antibody Leul 6. The modified Vh and Vk polypeptides differ from the native Vh or Vk regions of 2B8 and Leul6 by one or more amino acid residue substitution in the native sequence of the antibody Vh and/or Vk region. The amino acid residue substitution(s) in the Vh or Vk polypeptides afford a lower level of immunogenicity to the Cd-20 binding composition of the invention relative to the immunogenicity of the Vh or Vk regions of the native 2B8 and Leul 6 5 antibodies.
A CD20-binding polypeptide composition of the invention comprises at least one polypeptide segment selected from the group consisting of a polypeptide having the amino acid residue sequence of SEQ ID NO: 1 (VII of 2B8 antibody), but which includes at least one amino acid residue substitution in SEQ ID NO: 1 selected from the group consisting of V12K, A14P, M20V, 148T, A68T, Q82E, T87R, S91T, and T106W; a polypeptide having the amino acid residue sequence of SEQ ID NO: 4 (Vk of 2B8 antibody), but which includes at least one amino acid residue substitution in SEQ ID
NO: 4 selected from the group consisting of L1 I, S12T, S27T, V29A, G40T, V59S, S69T, L72M, R.76S, and V77L; a polypeptide having the amino acid residue sequence of SEQ
ID NO: 9 (Vh of Leul6 antibody), but which includes at least one amino acid residue substitution in SEQ ID NO: 9 selected from the group consisting of V12K, M20V, A68T, Q82E, T87R, S91T, D93V, and A114T; and a polypeptide having the amino acid residue sequence of SEQ ID NO: 11 (Vk of Leul6 antibody), but which includes at least one amino acid residue substitution in SEQ ED NO: 11 selected from the group consisting of L11I, SI2T, A59S, S69T, L72M, R76S, and Vnt.
As used herein and in the appended claims, an amino acid residue substitution is designated by listing the single letter code for the native amino acid residue in the sequence, followed by the position number of that residue (subscripted), followed by the single letter code for the amino acid residue that replaces the native amino acid.
Accordingly a substitution L1 II in SEQ ID NO: 4 means that the Leucine residue (L) at position 11 in SEQ ID NO: 4 (numbered from the N-terminus) is replaced (i.e., substituted) by an isoleucine (I).
Preferably the CD20-binding polypeptide compositions of the invention have a binding affinity for human CD20 antigen that is about equal to or stronger than the human CD20 binding affinity of monoclonal antibodies 2B8 or Leul6.
Preferred embodiments of the present invention comprise polypeptide segments such as a heavy chain termed herein VhC comprising the amino acid residue sequence of SEQ ID NO: 2:
QVQLQQ PGAEL KKPGASVKVS CKA.S GYT FT S YNMHWVKQT PGRGLEWTGAI YPG
NGDT S YNQKF KGKTTLTAD KS S S TAYMEL S SLRS EDTAVYYCAR S TYYGGDWYF
NVWGAGT TVTVSA;
a monoclonal antibody V-region heavy chain termed herein VhD comprising the amino acid residue sequence of SEQ ID NO: 3:
QVQLQQ P GAEL KKP GASVKVS CKAS GYTFT S YNMHWVKQT PGRGLEW I GAI YPG
NGDTSYNQKFKGKTTLTADKSSSTAYMELSSLRSEDTAVYYCARSTYYGGDWYF
NVWGAGTTVTVSA;
and a monoclonal antibody V-region heavy chain termed herein VhY comprising the amino acid residue sequence of SEQ ID NO: 10:
EVQL QQ SGAEL KKP GASVKVS CKAS GYT FT S YNMHWVKQT PGQGLEW I GAI YPG
NGDTSYNQKFKGKTTLTADKSSSTAYMELSSLRSEDTAVYYCARSNYYGSSYWFF
DVWGTGTTVTVSS.
Other preferred embodiments of the present invention comprise polypeptide segments such as a monoclonal antibody V-region light chain termed herein VkA
comprising the amino acid residue sequence of SEQ ID NO: 5:
QIVLSQSPAI ITAS PGEKVTMTCRASTSASYIHWFQQKPTSS PKPWIYATSNLASGVP
SRF SGSGSGTTYSMT I S SLEAEDAATYYCQQWTSNPPTFGGGTKLE I K;
a monoclonal antibody V-region light chain termed herein VkB comprising the amino acid residue sequence of SEQ ID NO: 6:
QIVLSQSPAI ITAS PGEKVTMTCRAST SVSYIHWFQQKPTS S PKPWIYATSNLASGVP
SRFSGSGSGTTYSMT I S SLEAEDAATYYCQQWTSNP PTFGGGTKLE I K;
a monoclonal antibody V-region light chain termed herein VkC comprising the amino acid residue sequence of SEQ ID NO: 7:
QIVLSQS PAI I TAS PGEKVTMTCRASTSVSYIHWFQQKPGSSPKPWIYATSNLASGV
PSRFSGSGSGTTYSMT I SSLEAEDAATYYCQQWTSNPPTFGGGTKLE I K;
a monoclonal antibody V-region light chain termed herein VkD comprising the amino acid residue sequence of SEQ ID NO: 8:
QIVLSQS PAI ITAS PGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGV
P SRFSGSGSGTTYSMT I SSLEAEDAATYYCQQWTSNPPTFGGGTKLE 1K;
and a monoclonal antibody V-region light chain termed herein VIcZ comprising the amino acid residue sequence of SEQ ID NO: 12:
DIVLTQSPAI I TAS PGEICVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASG
VPSRFSGSGSGTTYSMTIS SLEAEDAATYYCQQWSFNppTFGGGTKLE I K.
The present invention provides polypeptide compositions having therapeutic potential for the treatment of CD20 positive B-cell diseases in man. The CD20-binding polypeptide compositions of the invention can be in the form of intact antibodies, Fab fragments, or fusion proteins comprising whole antibodies and cytokines, fusion proteins of antibody fragments and cytokines, or other any other form that binds to CD20 antigen:
to The CD20-binging polypeptide compositions of the invention preferably include a combination of a heavy chain (Vh) anti-CD20 antibody variable region and a light chain (Vk) anti-CD20 antibody variable region, which together bind to CD20 antigen.
The Vh/Vk combinations can be configured as intact antibodies, Fab fragments, or fusion proteins comprising whole antibodies and cytokines, fusion proteins of antibody fragments and cytokines, or other any other configuration that binds to CD20 antigen.
In other preferred embodiments, the V regions are attached to each other through a polypeptide backbone. =
Preferably, the CD20-binding polypeptide compositions of the invention include Vh and Vk polypeptides configured as an intact antibody, which includes a light chain constant region, and heavy chain CH1, 0112, and CH3 domains. Alternatively, the Vh and/or Vk polypeptides can be configured into a Fab fragment or a "minibody"
having a 0113 domain but lacking a CH2 domain (see e.g., Wu et al., US 5,837,821).
Alternatively, the Vh and Vk polypeptides can be attached to each other through a linker to form an "single-chain Fv" antibody molecule. In one set of preferred embodiments, human constant regions are combined with the CD-20-binding Vh and Vk polypeptides. Such constant regions include those derived from IgA, IgD, IgM, IgE, or IgGl, IgG2, IgG3, or IgG4-type immunoglobulins. When a CH2 domain is included in the CD20-binding polypeptide composition, it is preferable to include a human IgG domain, such as an IgG1 CH2 domain. Other preferred embodiments lack the N-linked oligosaccharide glycosylation site found in the IgG CH2 domain.
The absence of the N-linked glycosylation site is preferably engineered by mutation of the relevant asparagine, serine or threonine, or adjacent amino acids, or to treat the CH2-containing protein with an enzyme such as PNGase F to remove the oligosaccharide.
Preferred combinations of Vh and Vk regions include a polypeptide composition comprising the heavy chain V-region VhC (SEQ ID NO: 2) and a the light chain VkA
(SEQ ID NO: 5); a polypeptide composition comprising the heavy chain V-region VhC (SEQ ID NO: 2) and a the light chain V- region VkB (SEQ ID NO: 6); a polypeptide composition comprising the heavy chain V-region VhC (SEQ ID NO: 2) and a the light chain V-region VkC (SEQ ID NO: 7); a polypeptide composition comprising the heavy chain V-region VhC (SEQ ID NO: 2) and a the light chain V-region VkD (SEQ ID NO: 8); a polypeptide composition comprising the heavy chain V-region VhD (SEQ ID NO: 3) and a the light chain V-region VkB (SEQ ID NO: 6);
a polypeptide composition comprising the heavy chain V-region VhD (SEQ ID NO: 3) and a the light chain V- region VkD (SEQ ID NO: 8); and a polypeptide composition comprising the heavy chain V-region VhY (SEQ ID NO: 10) and a the light chain V-region VkZ (SEQ ID NO: 12).
One preferred polypeptide composition of the invention includes a V-region heavy chain of antibody 2B8 (SEQ ID NO: 1), which includes one or more amino acid residue substitutions in SEQ ID NO: I selected from the group consisting of V12K, A14P, M20V, LaT, Adr, Q82E, T87R, S91T, and T106W.
Another preferred polypeptide composition comprises a V-region light chain of antibody 2B8 (SEQ ID NO: 4) modified to contain one or more amino acid residue substitutions in SEQ ID NO: 4 selected from the group consisting of Lill, SI2T, S27T, V29A, G40T, V59S, S69T, 1,72M, R76S, and V77L. Figure 16 illustrates the location of the preferred amino acid residue substitutions in bold face type.
Another preferred CD20-binding polypeptide composition of the invention includes a V-region heavy chain (Vh) of antibody Leul6 (SEQ ID NO: 9) modified to contain one or more substitutions in SEQ ID NO: 9 selected from the group consisting of V12K, M20V, Adr, Q82E, T87R, S91T, D93V, and A114T.
Yet another preferred polypeptide composition of the invention includes a V-region light chain (Vk) of antibody Leul6 (SEQ BD NO: 11) modified to contain one or more amino acid residue substitutions in SEQ ID NO: 11 selected from the group consisting of L11I, S12T, A59S, S59T, 1,72M, R76S, and V77L. Figure 16 illustrates the location of the preferred amino acid residue substitutions in bold face type.
The CD20-binding polypeptide compositions of the invention also include compositions having one or more anti-CD20 antibody heavy chain V region that comprises a polypeptide segment selected from the group consisting of SGAELKKPGAS (SEQ ID NO: 15), VSCKASGYT (SEQ ID NO: 16), LEWTGAIY (SEQ ID NO: 17), YNQKFKGKT (SEQ ID NO: 18), FKGKTTLTA (SEQ lD NO: 19), YMELSSLRS (SEQ ID NO: 20), SSLRSEDTAV (SEQ ID NO: 21), and DWGTGTTVT (SEQ ID NO: 22).
Similarly, the CD20-binding polypeptide compositions of the invention also include compositions having one or more anti-CD20 antibody light chain V region that comprises a polypeptide segment selected from the group consisting of IITASPGEKV (SEQ ID NO: 23), CRASTSASY (SEQ ID NO: 24), QQKPTSSP (SEQ ID NO: 25), LASGVPSRF (SEQ ID NO: 26), FSGSGSGTT (SEQ ID NO: 27), and YSMTISSLE (SEQ ID NO: 28).
The V region heavy chains and light chains can be configured, for example, as intact antibodies, antibody fragments, fusion proteins of an antibody with a cytokine, antibody fragment-cytokine fusion proteins, Fab molecules, single-chain Fv molecules, and minibodies. Preferably, the CD20-binding compositions are configured as intact antibodies, more preferably antibodies having human constant regions with and IgG
heavy chain constant region isotype, most preferably including an IgG1 heavy chain constant region and having intact effector functions such as antibody-dependent, cell-mediated cytotoxicity.
In another preferred embodiment the CD20-binding composition is configured as antibody-cytokine fusion protein, preferably including human constant regions with the isotype IgG1 and having intact effector functions such as antibody-dependent, cell-mediated cytotoxicity. In such preferred antibody-cytokine fusion proteins, it is useful to include an interleukin such as interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16 and IL-18, a hematopoietic factor such as granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), erythropoeitin, a tumor necrosis factors (TNF) such as TNFalpha, a lymphokine such as lymphotoxin, a regulator of metabolic processes such as leptin, an interferon such as interferon alpha, interferon beta, and interferon gamma, or a chemokine. Preferably, the antibody-cytokine fusion protein of the present invention 5 displays cytokine biological activity (e.g., stimulation of immune cells, such as T-cells or B-cells).
In a particular embodiment, the present invention relates to an anti-CD20 antibody molecule fused to IL-2 comprising: (i) a modified heavy chain variable region polypeptide having the amino acid residue sequence of SEQ ID NO: 9, but which 10 includes an amino acid residue substitution in said sequence selected from the group consisting of V12K, M20V, kir, Q82E, TuR, S91T, D93V, and A114T, (ii) a modified light chain variable region polypeptide having the amino acid residue sequence of SEQ ID
NO: 11, but which includes an amino acid residue substitution in said sequence selected from the group consisting of L111, S12T, k9S, S691-, I-72M, FZ76S, and V77L, and (iii) a human IgG heavy chain constant region and a human light chain constant region.
The polypeptide compositions of the present invention have a number of useful biological properties, including the ability to bind human 6-cells; the ability to bind to CD20 antigen; a reduced ability immune response relative to antibodies Leu16, 2B8, or 1H4; and activity against B cell proliferative disorders such as leukemias, lymphomas, rheumatoid arthritis, and other autoimmune diseases. Diagnostic uses for the CD2-binding polypeptide compositions of the present invention are also contemplated, in which composition can be a full length antibody, an antibody fragment (e.g. F(ab1)2), a radiolabelled antibody, an immobilized antibody, or an antibody conjugated with a heterologous compound. Such diagnostic method can be used to detect the presence of CD20-presenting cells, to purify or separate CD2-presenting cells, and the like.

10a The invention also provides pharmaceutical compositions comprising a CD20-binding composition as defined hereinabove together with a pharmaceutically acceptable carrier, diluent or excipient, as well as method of producing the polypeptide compositions.
In another embodiment, the present invention relates to the use of the anti-antibody fusion protein as described herein or the pharmaceutical composition as described herein for the treatment of CD20 expressing cancers.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 shows the native amino acid residue sequence, in single letter code, of the V-region heavy chain protein of the 268 antibody and the modified amino acid residue sequences VhC and VhD based on the native sequence.
FIGURE 2 shows the native amino acid residue sequence, in single letter code, of the V-region light chain protein of the 268 antibody and modified sequences VkA, VkB, VkC and VkD based on the native sequence.
FIGURE 3 shows the amino acid residue sequence, in single letter code, of the heavy chain variable regions of Leu16Vh and a modified Leu16Vh region designated VhY;
FIGURE 4 shows the amino acid residue sequence, in a single letter code, of the light chain variable regions of Leul6Vk and a modified Leu16Vk designated VhZ.
FIGURE 5 is a graphical representation of an antibody-dependent cellular cytotoxicity (ADCC) lysis assay described in Example 3; proteins tested were chimeric Leul antibody (chLeul6; filled triangles), a fusion protein of chLeul6 and IL2 (chLeul6-1L2; filled squares), LeuVhYNkZ-1L2 fusion protein (filled squares), chimeric FIGURE 6 illustrates the anti-CD20 binding activity of CD20- binding polyp eptide compositions and immunocytokines (ICs). Human Daudi lymphoma cells were FIGURE 7 illustrates antibody-dependent cell-mediated and complement-dependent cytotoxicity. Figures 7A and 7B represent independent ADCC tests compared to concentration (ng/ml).
FIGURE 7C shows CDC activity using 51Cr-labeled Daudi cells and human plasma as a source of complement, as described in Example 4. Incubation was for lhour using the same antibodies and immunocytokines. Antibodies tested were 2B8 (filled circles), FIGURE 8 illustrates the pharmaco-kinetics of epitope-depleted Leul 6 immunocytokines in mice, as described in Example 5. A time-concentration analysis was performed following an intravenous (i.v.) dose of each immunocytokine. Serum concentrations were determined by an ELISA that detects the intact forms for the native (solid squares) and de-glycosylated proteins (open circles). Mice were injected with 25 mg of the indicated immuncytokine and samples were taken by retro-orbital bleeding and assayed by capture with anti-human IgG H&L antisera and detection with an anti-human IL2 antibody.
Results are noulialized to the initial concentration in the serum of each mouse taken immediately after injection. X-axis: hours, Y-axis: % ID remaining FIGURE 9 shows anti-tumor model evaluations in SClD mice. SOD mice were injected i.v. with 2 x 106 CD20+ Daudi lymphoma cells followed by 5 consecutive daily injections of immunocytokines beginning on day 3 (Figure 9A) or day 7 (Figure 9B). The non-targeting control immunocytokine 425-IL2, specific for EGFR, was used at the high dose to illustrate the incomplete activity due to the altered half-life of IL-2. Treatments in Figure 9A were administered on days 3, 4, 5, 6, and 7 and included PBS only (filled diamonds); 425-I12 (dark, crossed X's); and Leul6VhY/VkZ-IL2 at daily doses of 5 micrograms (mcg) (light Xs), 10 mcg (filled triangles), and 20 mcg (filled squares). In Figure 9A, the latter three doses all showed complete protection of the mice, and the data points are superimposed.
Treatments in Figure 9B were administered on days 7, 8, 9, 10, and 11, and included PBS only (filled diamonds); and Leul6VhY/VkZ-1L2 at daily doses of 5 mcg (light Xs); and 20 mcg (filled squares). X-axis: days; Y-axis: % survival.
FIGURE 10 shows the results of anti-tumor model evaluations (2B8 vs. chLeu16-IL2) in SOD mice; SCID mice were injected with Daudi lymphoma cells and treated with the indicated antibody or immunocytokine beginning 7 days later, as described for Figure 9. Treatments included PBS only ( crossed Xs, on days 7-11); rituxirnab (C2B8; filled circles, 25 mg/kg on days 7,9 and 11); high dose Leul6VhY/VkZ-(large squares, 1 mg/kg on days 7-11); low dose Leul6VhY/VkZ-1L2 (small squares, 0.25 mg/kg on days 7-11). (A): healthy mice (B) survival of mice; X-axis:
days; Y-axis: % healthy mice (A), % survival of mice (B).
FIGURE 11 shows the results of anti-tumor model evaluations (chLeu16412 vs.
deimmunised (DI)-Leu16-IL2)in SCID mice; SCID mice were injected with Daudi lymphoma cells and treated with the indicated antibody or immunocytokine beginning 7 days later, as described for Figure 9. Treatments included PBS only ( crossed Xs, on days 7-11); rituximab (filled circles, 25 mg/kg on days 7, 9 and 11); high dose Leul6VhY/VkZ-1L2 (large squares, 1 mg/kg on days 7-11); low dose Leul6VhY/VkZ-1L2 (small squares, 0.25 mg/kg on days 7-11); high dose chLeu16-.
IL2 (large triangles, 1 mg/kg on days 7-11); and low dose chLeu16-1L2 (small triangles, 0.25 mg/kg on days 7-11). (A) healthy mice, (3) survival of mice.
X-axis: days; Y-axis: % healthy mice (A), % survival of mice (B).
FIGURE 12 illustrates that loss of ADCC activity has only a partial effect on anti-tumor activity mediated by Leul6VhYNKZ-1L2 (DI-Leu16-1L2 vs. deglycosyl. DI-Leu16-1L2). Two different doses of native and enzymatically deglycosylated Leul6VhY/VkZ-1L2 were used to treat SCID mice beginning 7 days after injection of Daudi lymphoma cells, as described for Figure 9. Treatments included PBS only (X, on days 7-11); rituximab (filled circles, 25 mg/kg on days 7, 9 and 11); high dose Leul6VhY/VkZ-1L2 (large triangles, 1 mg/kg on days 7-11); low dose Leul6VhYNkZ-1L2 (small triangles, 0.25 mg/kg on days 7-11); high dose deglycosylated Leul6VhY/VkZ-IL2 (large Xs with thick lines, 1 mg/kg on days 7-11);
and low dose de-glycosylated Leul6VhY/VkZ-1L2 (small Xs with thin lines, 0.25 mg/kg on days 7-11). (A) healthy mice, (B) survival of mice. X-axis: days; Y-axis: %
healthy mice (A), % survival of mice (B).
FIGURE 13 illustrates that antigen specificity is important for optimal anti-tumor activity. The role of tumor cell targeting was tested by comparing the activity of DI-Leu16-IL2 and another immunocytokine with binding specificity for EGFR, a molecule expressed at only low levels of Daudi lymphoma cells. Treatments included PBS only (X, on days 7-11); rituximab (filled diamonds, 25 mg/kg on days 7, 9 and 11); DI-leul6 antibody (open diamonds, 25 mg/kg on days 7, 9 and 11); medium dose DI-Leu16-IL2 (filled squares, 1 mg/kg on days 7-11); reduced dose DI-Leu16-1L2 (open circle, 1 mg/kg on day 7 and 10); low dose DI-Leu16-]L2 (open squares, 0.25 mg/kg on days 7-11); and medium dose anti-EGFR-1L2 (filled triangle, 1 mg/kg on days 7-11). Results were scored as disease free survival. X-axis: days; Y-axis: %
disease free.
FIGURE 14 illustrates that Leul6VhYNkZ-IL2 is more potent than higher doses of anti-CD20 antibody combined with free IL-2. Anti-tumor activities of medium dose Leul6VhY/VkZ-1L2 (filled squares, 20 mg/mouse on day 7-11) and the corresponding doses of the individual antibody and IL-2 components (open squares, 16.7 mg Leul6VhY, VICZ and 3.3 mg IL-2 by i.v. on day 7-11) were compared to high dose rituximab and subcutaneous (s.c.) IL-2 (open diamonds, 500 mg rituximab on day and 10 mg IL-2 on days 7,9 and 11), rituximab alone (filled diamond, 500 mg on day 7) or PBS control. X-axis: days;Y-axis: % disease free.
=
FIGURE 15 illustrates that the presence of normal anti-CD20+ B cells does not diminish the anti-tumor activity of Leul6VhY/VkZ-1L2. The effect of prior implantation of normal human CD20+ B cells was tested in the same SCID/Daudi lymphoma model as described in Methods. Groups of mice were treated with a single high dose of rituximab (diamonds, 25 mg/kg on day 7), Leul6VhY/VkZ-1L2 (squares, 1 mg/kg on days 11-15), the combination of both dosing regimens (triangles, rituximab on day 7 followed by Leul6VhY/VkZ-1L2 on days 11-15) or PBS alone on all dosing days. Half of the groups were injected i.v. with 4.5 x 106 PBMC on day 5 (open symbols) or received only PBS (filled symbols). B cell engrafillient was confirmed by measuring human antibody levels in the serum of all mice. X-axis: days;Y-axis:
%
disease free.
FIGURE 16 shows an alignment of the heavy chain and light chain V regions of the anti-CD20 antibodies 2B8, Leu16, and 1H4, as well as the epitope-depleted derivative V regions of 2B8 (VhC, VhD, VkA, VkB, VkC, and WD, and the epitope-depleted Leul6 V-regions VhY and 'VkZ.
FIGURE 17 shows a summary of all sequence identity numbers (SEQ ID Nos.: 1 ¨
43) as used in this application.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
As used herein an in the appended claims, the term "polypeptide composition"
and grammatical variations thereof refers to single chain polypeptides, as well as multiple chain polypeptides in which the polypeptide chains are chemically bound to each other, such as by one or more disulfide bonds between a cysteine residue in one chain and a cysteine residue on another chain, by an ester bond, an amide bond, or any other suitable linkage.
A CD20-binding polypeptide composition comprises at least one polypeptide segment selected from the group consisting of a modified heavy chain variable region polypeptide (Vh) having the amino acid residue sequence of SEQ ID NO: 1, but which includes at least one amino acid residue substitution in SEQ ID NO: 1 selected from the group consisting of V12K, A14P, M20V, 148T, A68T, Q82E, T87R, S91T, and T106W; a modified light chain variable region polypeptide (Vk) having the amino acid residue sequence of SEQ ID NO: 4, but which includes at least one amino acid residue substitution in SEQ ID NO: 4 selected from the group consisting of L11I, S12T, S27T, V29A, G4.0T, V59S, S69T, L72M, R.76S, and V77L; a modified Vh having the amino acid residue sequence of SEQ JD NO: 9, but which includes at least one amino acid residue substitution in SEQ ID NO: 9 selected from the group consisting of V12K, M20V, A68T, 5 Q82E, T87R, S9IT, D93V, and Ai14T; and a modified Vk having the amino acid residue sequence of SEQ ID NO: 11, but which includes at least one amino acid residue substitution in SEQ ID NO: 11 selected from the group consisting of L11I, S12T, A59S, S69T, 1,72M, R.76S, and V77L.
Preferably, the CD20-binding polypeptide composition includes at least one 10 polypeptide segment having an amino acid residue sequence selected from the group consisting of SEQ ID NO: 15, SEQ BD NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ
ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ lD NO: 23, SEQ
ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 15 and SEQ NO: 28.
In a preferred embodiment, the CD20-binding polypeptide composition comprises a modified heavy chain variable region polypeptide and a modified light chain variable region polypeptide. More preferably, the CD20- binding polypeptide composition is in the form of a chimeric antibody and also includes a human heavy chain constant region and a human light chain constant region. Preferably the human heavy chain constant region is an IgG constant region, more preferably an IgG1 constant region. The human light chain constant region preferably is a human kappa light chain constant region.
Another preferred embodiment of the CD20-binding polypeptide composition of the invention is a fusion protein comprising a polypeptide composition including modified heavy chain or light chain variable region segment, as described above, fused with a cytokine. Preferably the cytokine is IL2.
Another preferred embodiment of the CD20-binding polypeptide composition of the invention is an antibody molecule, such as a Fab antibody fragment, a single-chain Fv antibody fragment, or a minibody, which includes at least one modified Vh or modified Vk segment, as described above.

A particularly preferred CD20-binding polypeptide composition comprises at least one Vh polypeptide segment having an amino acid residue sequence selected from the group consisting of SEQ ID NO: 2, SEQ lD NO: 3 and SEQ ID NO: 10.
Another particularly preferred CD20-binding polypeptide composition comprises at least one Vk polypeptide segment having an amino acid residue sequence selected from the group consisting of SEQ ID NO:5 SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID
NO: 8, and SEQ ID NO: 12.
More preferably, the CD20-binding polypeptide composition comprises a Vh polypeptide segment having an amino acid residue sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 10; and a Vk polypeptide segment having an amino acid residue sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ
ID NO: 12.
Other preferred CD20-binding polypeptide compositions of the present invention comprise combinations of modified Vh and modified Vk segments selected from the group consisting of (a) a Vh segment having an amino acid residue sequence of SEQ ID NO: 2 and a Vk segment having an amino acid residue sequence of SEQ ID NO: 5;
(b) a Vh segment having an amino acid residue sequence of SEQ ID NO: 2 and a Vk segment having an amino acid residue sequence of SEQ ID NO: 6;
(c) a Vh segment having an amino acid residue sequence of SEQ lD NO: 2 and a Vk segment having an amino acid residue sequence of SEQ ID NO: 7;
(d) a Vh segment having an amino acid residue sequence of SEQ ID NO: 2 and a Vk segment having an amino acid residue sequence of SEQ ID NO: 8;
(e) a Vh segment having an amino acid residue sequence of SEQ lD NO: 3 and a Vk segment having an amino acid residue sequence of SEQ ID NO: 6;
(f) a Vh segment having an amino acid residue sequence of SEQ ID NO: 3 and a Vk segment having an amino acid residue sequence of SEQ ID NO: 8; and (g) a Vh segment having an amino acid residue sequence of SEQ ID NO: 10 and a Vk segment having an amino acid residue sequence of SEQ ID NO: 12.

= CA 02535515 2011-10-14 = 17 Another aspect of the present invention is a pharmaceutical composition comprising a CD20-binding polypeptide composition of the invention, together with a pharmaceutically acceptable carrier, excipient, diluent. The pharmaceutical composition can also include an additional pharmacologically effective drug.
The disclosed sequences have been derived from the analysis of the V-region sequences of the mouse monoclonal antibodies 2138 and Leul 6 referred to herein as the "!parental" antibodies and disclosed fully in respect of their useful in vitro and in vivo properties in US patent No. 5,736,137; US patent No. 5,776,456;US patent No.
6,399,061; US patent No. 6,455,043 and foreign equivalents, and in Wu et aL
(Protein Enginering (2001) 14:1025-1033) and references therein.
The sequence analysis so conducted was directed towards identification of the peptides contained within the sequence able to act a MHC class II ligands and therefore function as a T-cell epitope in man. An object of the present invention is to provide an improved version of the parental antibody. Thus knowledge of the positions of potential MHC class II epitopes is an important first step of the engineering process.
Epitope identification was conducted computationally, following the scheme outlined in detail in WO 02/069232. Following this first step, the inventors herein have surprisingly discovered sets of amino acid substitutions that collectively satisfy several different criteria with respect to making desired modifications to the parental antibodies.
The substitution sets within the V-regions of the parental antibody result in a polypeptide sequence with a reduced number of MHC class II ligands and accordingly a reduced propensity for the polypeptides to act as T-cell epitopes. Equally, the said substitution sets result in polypeptide sequences with the retained structural and functional properties of the parental antibody and this is true with respect to the especially desired dual properties of retained and stable expression within a host cell, and of particular importance, a retained ability for the polypeptide to bind the human CD20 antigen and especially bind human B-cells in vitro.
It is noteworthy that the Vh and Vk amino acid residue sequences of 288 and Leul 6, as well as the anti-CD20 antibody Bl, are quite similar, even though they are of independent origin. Without wishing to be bound by theory, an insight of the invention is as follows. The sequence CEPANPSEKNSPSTQYC (SEQ JD NO: 29) in human CD20, which is part of the short extracellular peptide of CD20 and which is flanked by cysteines that are likely to form a disulfide bond, corresponds to the sequence CEPSNSSEKNSPSTQYC (SEQ ID NO: 38) in mouse CD20. The mouse NSS
sequence is likely to be an N-linked glycosylation site, while the corresponding NPS
sequence in humans is not likely to be an N-linked glycosylation site. This difference between the mouse and human CD20 sequences would reveal, from the vantage point of the mouse immune system, an epitope in the human sequence. Thus, all of the mouse antibodies generated against human CD20 may be selected to recognize a very narrowly defined epitope, and there may be one optimal set of mouse V regions to do this.
The invention thus provides a set of reduced-immunogenicity consensus V region sequences that are important for recognition of human CD20. For example, in the heavy chain, the sequence segment VSCKASGYT (SEQ ID NO: 16) in the modified heavy chains of the invention is useful in the context of an antibody that binds to CD20; specifically, this segment is less immunogenic than the corresponding segment, MSCKASGYT (SEQ ID NO: 30), from the 2B8 and Leul6 antibodies. Similarly, the sequences YNQKFKGKT (SEQ ID NO: 18), FKGKTTLTA (SEQ ID NO: 19), and YMELSSLRS (SEQ ID NO: 20) are useful in the context of CD20 antibodies, and are less immunogenic than the corresponding sequences, YNQKFKGKA (SEQ DD NO:
31), FKGKATLTA (SEQ ID NO: 32), and YMQLSSLRS (SEQ ID NO: 33), that are found in the parental 2B8 and Leul6 antibodies.
Considering the light chain, the sequence segment IITASPGEKV (SEQ ED NO: 23) in the modified light chains of the invention is useful in the context of an antibody that binds to CD20; specifically, this segment is less immunogenic than the corresponding segment, ILSASPGEKV (SEQ ID NO: 34), from the parental 2B8 and Leul6 antibodies. Similarly, the sequences LASGVPSRF (SEQ ID NO: 26), FSGSGSGTT
(SEQ ED NO: 27), and YSMTISSLE (SEQ ID NO: 28) are useful in the context of CD20 antibodies, and are less immunogenic than the corresponding sequences, LASGVPVARF (SEQ ID NO: 35), FSGSGSGTS (SEQ ID NO: 36), and YSLTISRVE
(SEQ ID NO: 37), that are found in the parental 2B8 and Leul6 antibodies.
Reference to "substantially non-immunogenic" or "reduced immunogenic potential,"
as used herein and in the appended claims, includes reduced immunogenicity compared to a counterpart or "parent" antibody, i.e. a non-modified murine or chimeric monoclonal antibody such as 2B8 or Leu16. The term "immunogenicity" includes an ability to provoke, induce or otherwise facilitate a humoral and or T-cell mediated response in a host animal and in particular where the "host animal" is a human.
The term "antibody molecule" refers to a polypeptide of the immunoglobulin family, including a whole antibody, that is capable of combining, interacting or otherwise associating with an antigen.
The tem]. "antigen" is used herein to refer to a substance that is capable of interacting with the antibody molecule and in the context of the present invention is meant to be CD20. The CD20 of the present invention is human CD20 or any CD20 representing an antigen for antibody 2B8. The CD20 may be a soluble CD20 derivative or membrane associated CD20.
The term "immunoglobulin" is used herein to refer to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized immunoglobulin genes include IgGl, IgG2, IgG3, IgG4 constant region genes and in nature multiple immunoglobulin variable region genes. One natural form of immunoglobulin is a tetramer comprising two identical pairs in which each pair has one light chain and one heavy chain. In each pair the heavy and light chain variable regions together provide the binding surface capable of interacting with the antigen.
The term Vh is used herein to refer to the heavy chain variable region, and the term Vk is used herein to refer to the light chain variable region and in this instance in common with numerous monoclonal antibodies the light chain is a "kappa" (k) type chain.
The V-region includes amino acid residues from a "complementarity determining region" or "CDR" (i.e. at about amino acid residues 24-34 (L1), 50-56 (L2) and (L3) in the light chain variable domain and at about amino acid residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain as defined by Kabat et al. (Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
Alternative definitions of the CDRs are also recognized in the art for example according to the scheme of Chothia (Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917) in which case the CDRs are found at about amino acid residues 26-32 (L1), 50-52 (L2) and 91-(L3) in the light chain, and at about amino acid residues 26-32 (H1), 53-55 (H2) and 96-101(113) in the heavy chain. "Framework" or "FR" residues are those V-region residues other than the CDR residues as herein defined.
As used herein, Vh means a polypeptide that is about 110 to 125 amino acid residues 5 in length, the sequence of which corresponds to any of the specified Vh chains herein which in combination with a Vk are capable of binding human CD20. Similarly, Vk means a polypeptide that is about 95-130 amino acid residues in length, the sequence of which con-esponds to any of the specified Vk chains herein which in combination with a Vh are capable of binding human CD20. Full-length immunoglobulin heavy 10 chains are about 50 kDa molecular weight and are encoded by a Vh gene at the N-terminus and one of the constant region genes at the C-terminus. Similarly, full-length light chains are about 25 kDa molecular weight and are encoded by a V-region gene at the N-terminus and a constant region gene at the C-terminus.
15 In addition to whole a antibody (a tetramer), immunoglobulins may exist in a number of other forms derived by application of recombinant DNA techniques or protein biochemistry. These forms include for example Fv, Fab, Fab' and (Fab)2 molecules and could all contain any of the Vh or Vk sequences of the present invention.
A
further example may include a "bi-specific" antibody, that is comprising a VhNk 20 combination of the present invention in combination with a second Vh/Vk combination with a different antigen specificity.
The tenn "T-cell epitope," as used herein and in the appended claims, means an amino acid sequence which is able to bind MEC class II, able to stimulate T-cells and / or also to bind (without necessarily measurably activating) T-cells in complex with MHC
class II.
The term "peptide" as used herein and in the appended claims, is a compound that includes two or more amino acid residues. The amino acid residues are linked together by a peptide bond (defined herein below). There are 20 different common, naturally occurring amino acids involved in the biological production of peptides, and any number of them may be linked in any order to form a peptide chain or ring. The naturally occurring amino acids employed in the biological production of peptides all have the L-configuration. Synthetic peptides can also be prepared employing conventional synthetic methods, utilizing L-amino acids as well as D-amino acids, or various combinations of amino acids of the two different configurations. Some peptides contain only a few amino acid residues. Short peptides, e.g., having less than ten amino acid residues, are sometimes referred to as "oligopeptides". Other peptides contain a large number of amino acid residues, e.g. up to 100 or more, and are referred to as "polypeptides".
By convention, a "polypeptide" may be considered as any peptide chain containing three or more amino acid residues, whereas an "oligopeptide" is usually considered as a particular type of "short" polypeptide. Thus, as used herein, it is understood that any reference to a "polypeptide" herein also includes an oligopeptide. Further, any reference to a "peptide" includes polypeptides, oligopeptides, and proteins.
Each different arrangement of amino acid residues forms different polypeptides or proteins.
The number of polypeptides¨and hence the number of different proteins¨that can be formed is practically unlimited.
The tem]. "peptide composition" means a composition which at least comprises one polypeptide deriving from an anti-CD20 antibody, preferably 2B8 and Leu16. The polypeptide my be exist as single chain (e.g. heavy chain or part thereof, light chain or part thereof, or single-chain Fv) or in a dimeric form. The dimeric form may be a whole antibody, a Fab fragment, a minibody, or any two-chain molecule, wherein the single chains are linked by a mere chemical bond. The composition may comprise several single chain polypeptides only, which are not linked by a chemical or a cysteine bond.
The invention also relates to an anti-CD20 monoclonal antibody in which substitutions of at least one amino acid residue have been made at positions within the V-regions of the molecule to result in a substantial reduction in activity of or elimination of one or more potential T-cell epitopes from the protein. It is most preferred to provide modified antibody molecules in which amino acid modification (e.g. a substitution) is conducted within the most immunogenic regions of the parent molecule. The major preferred embodiments of the present invention comprise modified antibody molecules for which any of the MHC class II ligands are altered such as to eliminate binding or otherwise reduce the numbers of MHC allotypes to which the peptide can bind.
For the elimination of T-cell epitopes, amino acid substitutions are made at appropriate points within the peptide sequence predicted to achieve substantial reduction or elimination of the activity of the T-cell epitope. In practice an appropriate point will preferably equate to in amino acid residue binding within one of the pockets provided within the MHC class II binding groove.
It is most preferred to alter binding within the first pocket of the cleft at the so-called P1 or P1 anchor position of the peptide. The quality of binding interaction between the P1 anchor residue of the peptide and the first pocket of the MHC class II
binding groove is recognized as being a major determinant of overall binding affinity for the whole peptide. An appropriate substitution at this position of the peptide is for a residue less readily accommodated within the pocket, for example, substitution to a more hydrophilic residue. Amino acid residues in the peptide at positions equating to binding within other pocket regions within the MHC binding cleft are also considered and fall under the scope of the present.
It is understood that single amino acid substitutions within a given potential T cell epitope are the most preferred route by which the epitope may be eliminated.
Combinations of substitution within a single epitope may be contemplated, and for example, can be particularly appropriate where individually defined epitopes are in overlap with each other. Moreover, amino acid substitutions either singly within a given epitope, or in combination within a single epitope, may be made at positions not equating to the "pocket residues" with respect to the MHC class II binding groove, but at any point within the peptide sequence. All such substitutions fall within the scope of the present.
An important and significant feature of the modified antibody molecules of the present invention is that they retain the functional activities of the non-modified parental antibody. It is therefore particularly desired to produce modified antibodies or modified antibody molecules in which all of the beneficial technical features associated with the therapeutic efficacy of the parental non-modified antibody are exhibited. This is pertinent to the contemplated utility of the invention, namely to provide a composition with therapeutic efficacy in a number of important diseases in man including especially B-cell lymphoma and other B-cell mediated pathologies.
Such a therapeutic is a preferred embodiment of the present invention.
Accordingly, the modified antibody molecule of the present invention exhibits an affinity for its target antigen that is similar to the affinity exhibited by the parental antibody. The antibody molecule therefore recognizes CD20 positive human B-cells.
The therapeutic efficacy of the parental molecule is considered to be mediated by the ability of the antibody to induce antibody-dependent cellular cytoxicity (ADCC).
Critical to this activity is the ability of the antibody constant region (i.e.
the Fe domain) to bind human serum complement component Clq. ADCC and Clq binding is mediated by the constant region domain of whole antibody molecules, and the present invention contemplates production of whole antibody molecules comprising a human Fe compatible with ADCC induction. Such constant regions are most preferably IgG1 heavy chains in combination with human kappa light (e.g. Km3) chains.
In as far as this invention relates to modified anti-CD20 antibody molecules, compositions containing modified antibodies or fragments of modified antibodies and related compositions are within the scope of the invention. The invention therefore contemplates the use and generation of antibody fragments including for example Fv, Fab, Fab' and F(ab)2 fragments. Such fragments may be prepared by standard methods (for example; Coligan et al. (Current Protocols in Immunology, John Wiley &
Sons 1991-1997). The present invention also contemplates the various recombinant forms of antibody molecule derived molecular species well known in the art. Such species include stabilised Fv fragments including single chain Fv forms (e.g. scFv) comprising a peptide linker joining the Vh and Vk domains, or an Fv stabilized by inter-chain disulfide linkage (dsFv) and which contain additional cysteine residues engineered to facilitate the conjoining of the Vh and Vk domains. Equally, other compositions are familiar in the art and could include species referred to as "minibodies"; and single variable domain "dAbs". Other species still may incorporate means for increasing the valency of the modified antibody V-region domain, i.e. species having multiple antigen binding sites for example by the engineering of dimerisation domains (e.g.
"leucine zippers") or also chemical modification strategies.
In another aspect, the invention relates to fusion proteins in which the anti-regions of the invention are coupled to a non-immunoglobulin fusion partner protein, such as an anticancer protein. Examples of such proteins include toxins, such as Pseudomonas exotoxin; enzymes, such as bacterial proteases for antibody-dependent prodrug therapy ("ADEPT"), or cytokines. Particularly useful cytokines are wild-type and mutant versions of IL-2, I1-12, fused forms of 1L-2 and 11-12, and other = CA 02535515 2011-10-14 interleulcins, interferons, and tumor necrosis factors. Gullies and co-workers (US5,150,650, W098/25978, W001/10912, W002/72605, W003/48334), Epstein (WO 03/15697), and flalin et al. (Cancer Res. (2003) 63:3202-10) have described a number of configurations of anti-cancer antibody V regions and cytokines. The immunoglobulin constant regions of fusion proteins comprising the V regions of the present invention may be further modified by mutations, such as mutations affecting complement fixation, mutations affecting Fc receptor binding, mutations affecting FcRn binding, and mutations affecting the serum half-life of the fusion protein;
examples of such mutations are described in W009943713 and W001/58957.
In a further aspect, the present invention relates to isolated nucleic acids encoding CD20-binding polypeptide compositions, such as nucleic acids that encode a modified anti-CD20 antibody molecule, a modified heavy chain variable region polypeptide, a modified light chain variable region polypeptide, anti-CD20 antibody fusion proteins, and the like. The DNA sequence of the polynucleotide encoding the light chain of Leul6 is shown in FIG. 17 (SEQ ID NO: 39). The DNA sequence encoding a preferred epitope-depleted version of the light chain of Leul6 (i.e., including Vla and a human constant region) is shown in FIG. 17 as SEQ ID NO: 40. The DNA
sequence of the polynucleotide encoding the heavy chain of Leul6 is shown in FIG. 17 (SEQ ID
NO: 41). The DNA sequence encoding a preferred epitope-depleted version of the heavy chain of Leul6 (i.e., including VhY and a human constant region) is shown in FIG. 17 as SEQ ID NO: 42. The DNA sequence encoding a preferred epitope-depleted version of the heavy chain of Leul6 fused to IL-2 (i.e., including VhY, a human constant region, and IL-2 bound to the C-terminus of the constant region) is shown in , FIG. 17 as SEQ NO: 43.
Yet another aspect the present invention relates to methods for therapeutic treatment of humans using the CD20-binding polypeptide compositions of the invention. The Examples illustrate how the polypeptide compositions of the invention have been used to treat human cancer cells in mouse models, and the results obtained below are generally illustrative of strategies that would be used to treat human cancers such as B
cell lymphoma and other cancers expressing CD20.
=

The CD20-binding polypeptide compositions of the invention, such as anti-CD90-fusion proteins, are used as follows: A patient suffering from a CD20-expressing cancer, such as B cell lymphoma, is administered a polypeptide composition of the invention. The preferred route of administration is intravenous or subcutaneous 5 injection, but intramuscular, intraperitoneal, intrademial, or other routes of injection are also possible. Administration by inhalation, orally, or by suppositories is also possible, as are other routes of administration. Administration is preferably in a four-week cycle of three times per week, followed by no treatment for the next three weeks.
Treatments can be more or less frequent depending on the pharmacokinetic behavior of 10 the fusion protein in a given individual. The preferred dosage for an adult of about 70 kilograms is in the range of about 1 to about 100 milligrams per dose, with a preferred range of about 4 to about 20 milligrams per dose. The most preferred dose is about 10 milligrams for a 70 kg adult treated once per month. Patients are monitored for a response according to standard procedures.
EXAMPLES
The Examples describe certain particular methods for making proteins of the invention, but those skilled in the art of protein expression will recognize that a wide variety of well-blown techniques may be used to make proteins of the invention. For example, the proteins of the invention are preferably made in eukaryotic cells such as mammalian cells, such as NS/0 cells, BHK cells, CHO cells, 293 cells, or PERC6 cells.
An protein of the invention may, for example, be purified using, in sequence, some or * =
all of the following steps: Abx Mixed Resin column chromatography, recombinant Protein A chromatography, and Q Sepharose column chromatography, followed by Pellicon 2 angential flow diafiltration for buffer exchange into formulation buffer.
Virus inactivation and removal steps are interdigitated into these steps. The virus inactivation and removal steps are not necessary for purificationper se, but are used to satisfy regulatory considerations. In a further aspect the present invention relates to methods for therapeutic treatment of humans using the modified anti-CD20 antibody molecules. The Examples illustrate how the proteins of the invention have been used to treat human cancer cells in mouse models, and the results obtained below are generally . illustrative of strategies that would be used to treat human cancers such as B cell lymphoma and other cancers expressing CD20.
* Trade-mark = CA 02535515 2011-10-14 The fusion proteins of the invention are used as follows. A patient suffering from a CD20-expressing cancer, such as B cell lymphoma, is treated. The preferred route of administration is intravenous or subcutaneous injection, but intramuscular, intraperitoneal, intradermal, or other routes of injection are also possible.
Administration by inhalation, orally, or by suppositories is also possible, as are other routes of administration. Administration is preferably in a four-week cycle of three times per week, followed by no treatment for the next three weeks, but may be more or less frequent depending on the pharmacokinetic behavior of the anti-CD20-fusion protein in a given individual. Dosing for an adult of about 70 kilograms is in the range of about 1 to 100 milligrams per dose, with a preferred range of about 4 to 20 milligrams per dose. The most preferred dose is about 10 milligrams for a 70 kg adult treated once per month. Patients are monitored for a response according to standard procedures.
Example 1. Methods and reagents for expressing DeImmunised Leul6 antibodies.
1A: Cell culture and transfectio: In order to obtain stably transfected clones, plasmid DNA was introduced into the mouse myeloma NS/0 cells by electroporationi.
About 5 x 106 cells were washed once and re-suspended with phosphate buffer solution (PBS).
Ten [ig of linearized plasmid DNA was then incubated with the cells in a Gene Pulser =vette (0.4 cm electrode gap, BioRad) for 10 minutes on ice. Electroporation was performed using a Gene Pulser (BioRad) with settings at 0.25 V and 500 F.
Cells were allowed to recover for 10 minutes on ice, after which they were re-suspended in growth medium and then plated onto 96-well plates. Stably transfected clones were selected by growth in the presence of 100 nM methotrexate (MTX), which was introduced two days post-transfection. The cells were fed every 3 days for 2 or 3 more times and MTX-resistant clones appeared in 2 to 3 weeks. Supernatants from clones were assayed by anti-human FC ELISA and analytical high-pressure liquid chromatography (IIPLC), to identify high producers (Gilles et al. (1989) J.
Immunol.
Methods 125:191). High producing clones (yielding about100 lig of purified protein per ml of cell culture supernatant in medium scale cell cultures) were isolated and propagated in growth medium containing 100 mM MTX. .
1B: ELISAs: ELISAs.were used to determine the concentrations of protein products in the supernatants of MTX-resistant clones. The anti-h-uFc ELISA was used to measure * Trade-mark _ the amount of human Fe-containing proteins. The anti hu-Fc ELISA is described in detail below.
A. Coating Plates: ELISA plates were coated with AFFINIPURBTM goat anti-human IgG (H+L) (Jackson Immuno Research) at 5 jig/m1 in PBS and 100 0/well in 96-well plates (Nunc Immuno Plate Maxisorp). Coated plates were covered and incubated at 4(C overnight. Plates were then washed 4 times with 0.05% Tween (Tween 20) in PBS
and blocked with 1% bovine serum albumin (BSA)/ 1% goat serum in PBS, 200 id/well. After incubation with the blocking buffer at 37 C for 2 hours, the plates were washed 4 times with 0.05% Tween and tapped dry on paper towels.
to B. Incubation with test samples and secondaly antibody: Test samples were diluted to the proper concentrations in sample buffer, which contained 1% BSA/ 1% goat serum/ 0.05% Tween in PBS. A standard curve was prepared with a chimeric antibody (bearing a human Fe), the concentration of which was known. To prepare a standard curve, serial dilutions are made in the sample buffer to give a standard curve ranging from 125 ng/ml to 3.9 ng/ml. The diluted samples and standards were added to the plate, 100 ill/well and the plate incubated at 37 C for 2 hours.
After incubation, the plate was washed 8 times with 0.05% Tween in PBS. To each well was then added 100 p.1 of the secondary antibody, the horse radish peroxidase (HRP)-conjugated anti-human IgG (Jackson Immuno Research), diluted around 1:120,000 in the sample buffer. The exact dilution of the secondary antibody had to be determined separately for each lot of the HRP-conjugated anti-human IgG. After incubation at 37 C for 2 hours, the plate was then washed 8 times with 0.05%
Tween in PBS.
C. Development: The ready-made substrate solution (TMB Substrate, BioFX
Laboratories, MD) was added to the plate at 100 td/well and the color allowed to develop at room temperature for 10 minutes. The reaction was stopped by adding IN
HC1, 100 pi/well. The plate was read by a plate reader which was set to a wavelength of 450 mn.
IC: Analytical HPLC based method for determining protein concentration:
Protein concentrations were also determined using analytical HPLC. A standard curve for protein concentration over the range 0.78 to 50.0 ilg/m1 was determined using a pH-based elution protocol with a POROS column (Perceptive Biosystems) on an Agilent 1100 HPLC system, and with the KS-1L2 imrnunoconjugate as a standard.
* Trade-mark Example 2. Determination of the relative binding affinity of the epitope-depleted Len-16 antibody-1L2 fusion protein for Daudi tumor cells presenting the CD20 antigen. The binding of the epitope-depleted Leul6-IL2 fusion proteinto Daudi lyrnphoma cells bearing the CD20 antigen, was compared with the binding of other known anti-CD20 antibodies using flow cytometry analysis. Approximately 106 Daudi cells were used with various concentrations of antibody in a 100 pi volume, for each sample tested. The analysis showed that the modified antibody-1L2 fusion protein with VHY and VKZ variable regions bound to CD20-expressing cells at least as well as the corresponding chimeric Leul6-1L2 fusion protein. This shows that the mutations introduced into the Leul6 V regions to generate VHY and VKZ did not interfere with antigen binding. The antibody-1L2 fusion protein with VHY and VKZ variable regions also compared favorably to RITUXAN (C2B8), as well as the 2B8-1L2 fusion protein containing the murine 2B8 V regions. Data in Table 1 below shows the mean fluorescence intensity, as measured in a flow cytometer, of Daudi cells exposed to different concentrations of antibody or antibody-1L2 fusion protein; this data represents a typical set of experimental results.
Table 1 Antibody Concentration (g/ml) Antibody 10 5 2.5 1.25 0.625 0.313 0.156 0.078 2B8-1L2 857.32 720.02 512.81 369.-04 229.45 147.26- 85.32 52.13 Ab(VhY/V1a-1L2 987.54 848.56 677.11 539.35 336.41 219.48 124.33 74.77 chLeu16-1L2 789.20 665.20 489.04 363.00 219.06 131.92 78.24 52.31 997.18 816.47 647.43 491.38 311.52 215.34 130.70 86.48 Example 3. Antibody-dependent, cell-mediated cytotoxicity (AD CC) driven by anti-CD20-1L2 fusion proteins carrying mutations that reduce immunogenicity.
NS/0 cells that express CD20 were exposed to various concentrations of antibody or antibody-1L2 fusion protein and tested for ADCC lysis according to standard procedures. A typical data set is shown in FIGURE 5. Proteins tested were as follows:
chLeul6 is a chimeric antibody consisting of the Leul6 mouse V regions and human IgG1 constant regions; chLeu16-1L2 is a chimeric antibody consisting of the Leul6 mouse V regions and human IgG1 constant regions, with a human IL-2 moiety fused to the C-terminus of the antibody heavy chain; Ab(VhY/VkZ)-1L2 is identical to chLeu16-1L2 except that the 'VhHY and VkZ variable domains listed in FIGURES 3 and 4 were present instead of the murine Leul6 V regions; 2B8 is, in this figure, a chimeric antibody consisting of the 2B8 mouse V regions and human IgG1 constant regions; 2B8-1L2 is, in this figure, a chimeric antibody consisting of the 2B8 mouse V
regions and human IgG1 constant regions, with a human IL-2 moiety fused to the C-terminus of the antibody heavy chain; and KS-1L2 an anti-EpCAM antibody, with a human IL-2 moiety fused to the C-terminus of the antibody heavy chain. The KS-protein serves as a negative control. Certain of the antibody-1L2 fusion proteins had mutations of the C-terminal heavy chain amino acids as described by Gillies et al.
(W002/66514); these mutations had no effect on the ADCC data.
The data indicate that a fusion protein bearing the VhY and Vkl variable regions was as active as similar molecules carrying CD20-binding murine V regions in stimulating ADCC. EL2 activity was determined by several different cell assays. The results are presented in Table 2 below.
Table 2 T Cell T Cell +IL2 R
CTLL-2 HU PBMC Kit-225 TF-1 Avg.ED50 Avg.ED50 Avg.ED50 Avg.ED5o (ng/ml) (ng/ml) (ng/ml) (ng/ml) KS IL2 1.71 2.09 0.08 0.51 R&D Hu IL2 0.65 1.51 0.07 0.71 ChLeu16-1L2 0.06 1.15 Dl-Leu16-1L2 1.94 2.03 0.05 1.14 D1-Leu-16-1L2 3.30 3.42 0.09 1.99 Degly It has also been found that IL-2 based immunocytokines targeting CD20 are highly efficacious in SOD mouse models of well established lymphoma, at least in the absence of normal human B cells expressing the same surface antigen, and that the use of immunocytokines was far more effective than that of the naked antibody in extending the survival of mice with disseminated disease, despite the lack of functional T cells, cells that have been identified in many pre-clinical studies to be the primary effectors of immunocytokine anti-tumor activity.

Example 4. Complement-dependent cytotoxic4 (CDC) driven by anti-CD20-1L2 fusion proteins carrying mutations that reduce immunogenicity.
To determine CDC activity of antibodies and fusion proteins of the invention, 51Cr-labeled Daudi cells were incubated for 1 hour with human plasma (diluted 1 in 8) as a 5 source of complement. Percentage of specific lysis was calculated by subtracting the background radioactivity from the experimental values, dividing by the total releasable radioactivity obtained by detergent lysis, and multiplying by 100.
A comparison of the CDC activities of the chimeric Leu-16 antibody and the corresponding Leul6 antibody with epitope-depleted V regions indicates that the 10 activities of these two antibodies were essentially identical. A similar comparison of the CDC activities of the chimeric Leu-16 antibody-1L2 fusion and the corresponding Leul 6 antibody-1L2 fusion with epitope-depleted V regions indicates that the activities of these two fusion proteins were also essentially identical.
In contrast to ADCC, CDC was somewhat reduced as a consequence of fusing IL-2 to 15 the C-terminus of the H chain (Figure 2B). A similar effect was reported earlier with an anti-GD2 immunocytokine (Gillies SD, et al. Cancer Res. (1999);59:2159-2166.).
Typical results are shown in Figure 7C.
Example 5. Pharmacokinetic profile of an epitope-depleted Leu16-IL2 fusion protein.
20 In a particularly useful Leul6VhY/VkZ-IL2 construct, FcR binding and ADCC
activity were maintained by using a Cgl heavy chain isotype but have incorporated a modified junction region between the H chain and IL-2 in order to reduce intracellular proteolysis (W001/58957). Specifically, a Lysine to Alanine change corresponding to the C-terminal amino acid of the antibody heavy chain was used. The resulting protein 25 had a favorable pharmacokinetic profile following i.v. administration, especially during the distribution a phase (Figure 8). The effect of FcR binding on the pharmacokinetic profile was examined by testing the enzymatically de-glycosylated Leul6VhY/VkZ (Lys-Ala)-1L2 in the same experiment. The results indicate that loss of FcR binding improved the pharmacokinetic behavior somewhat. Depending on the 30 application and the desired frequency of administration, it may be preferable to use a Leul6VhY/VkZ-1L2 molecule with the N-linked glycosylation site in the CH2 domain, which has ADCC activity but a relatively shorter serum half-life, or a Leul6VhY/VkZ-1L2 molecule lacking the N-linked glycosylation site in the CH2 domain, which has no ADCC activity but a relatively longer serum half-life.

Example 6. Efficacy profile of an epitope-depleted Leu16-1L2 fusion protein.
SCID mice were injected i.v. with 5 x 106 CD20+ Daudi lymphoma cells (day 0) followed by i.v. injection of immunocytokines (5 daily doses of 5 or 20 mg) or control antibody (500 mg every other day for a total of 3 doses) beginning on day 7. A
low-targeting control immunocytokine 425-1L2, specific for EGFR (control), was used at the high dose to demonstrate activity due primarily to the altered half-life of IL-2.
Results were recorded as general health, e.g. paralysis, which preceded death by 10-14 days, and survival of mice. Figures 9-12 show typical results. Data in Figures are from a single, large experiment, but the data are presented in different figures for ease of viewing. In the first anti-tumor experiments Daudi cells were injected i.v. into SCID mice resulting in extensive disseminated disease leading to paralysis of all mice by day 30. Treatment was delayed until day 7 to ensure the tumor cells had fully engrafted. We compared low and moderate doses of both the chimeric and Leul6VhYNkZ-1L2 immunocytokines to high dose rituximab using 5 daily doses of the immunocytokines and 3 alternate day doses of the antibody. This schedule was chosen due to the much longer circulating half-life of rituximab (several days) compared to the immunocytokines (about 8 hours). Under these conditions, rituximab (25 mg/kg x 3) extended the 50% survival of tumor bearing mice from 39 to 56 days, relative to the PBS control (Figure 4). The low dose chimeric and Leul6VhY/VkZ-groups (0.25 mg/kg x 5) had similar survival curves (50% survival at 64 days) as the high-dose rituximab. The groups treated with the higher doses of the immunocytokines (1 mg/kg x 5) showed a dramatic increase in survival with no mouse deaths in the Leul6VhY/V1a-1L2 group at the termination of the experiment (day 110) and only 1 of 8 mice dead in the chLeu16-1L2 group. Thus, the epitope-depleted V
regions of the Leul6 antibody were as effective as those of the murine Leul6 antibody, in the context of an IL-2 based immunocytokine, for the treatment of disseminated lymphoma in SCID mice. In the same experiment the contribution of antibody effector function on anti-tumor activity was also tested using the enzymatically de-glycosylated Leul6VhY/V1a-1L2, which was shown above to have lost ADCC activity (Figure 7B).
As shown in Figure 12, a significant portion of the anti-tumor activity was preserved despite the loss of ADCC activity. At later time points, a marked difference between the intact and de-glycosylated forms was observed in the higher dose groups.
Therefore, while ADCC appears to play a role in this model, a good deal of the anti-tumor activity in this model can be attributed to targeted delivery of IL-2 to the tumor alone. To address the importance of actual binding of an epitope-depleted immunocytokine to a target cell, as opposed to an effect of simply extending the serum half-life of IL-2, a control IL-2 immunocytokine targeting EGFR (Cruz et al. J

Biotechnol. (2002) 26;96:169-183), which is expressed at only very low levels (Figure 6B) on this cell line, was tested. It was found that even the higher dose (1 mg/kg) given 5 consecutive days had far less anti-tumor activity in this model than the same dose of the immunocytokines targeting CD20 (Figure 13). The EGFR-targeted immunocytokine also had significantly less activity than the same dose of Leul6VhY/VKkIL2 given only twice, three days apart (days 7 and 10), or at a four-fold lower dose given over 5 days. These results demonstrate the importance of specific tumor cell targeting for anti-tumor activity.
Anti-tumor activity of separate antibody and IL-2 components. Clinical trials combining rituximab and IL-2 have shown increased response rates (Friedberg et al.
Br. 1 Haernatol. (2002); 117: 828-834). This combination was tested in the same Daudi lymphoma model using two different approaches and compared to treatment with Leul6VhY/VkZ-1L2. ha the first case, animals were dosed i.v. for 5 consecutive days with the equivalent molar amounts of Leul6VhYNkZ antibody and IL-2 contained in 20 mg of Leul6VhY/VkZ-1L2. In the second case, 25 mg/kg of rituximab and 10 mcg of IL-2 were given s.c. every other day for 3 doses. This latter dosing regimen would ensure high levels of antibody as well as a sustained IL-2 activation due to the depot effect of s.c. administration. Results indicated that the two combination protocols resulted in roughly the same degree of anti-tumor activity, with 50% survival of 63 days (Figure 14). Treatment with the equivalent amount of Leul6VhY/VkZ-1L2 immunocytokine, used in the low dose combination group, resulted in long-term survival of all mice. This is particularly noteworthy since the groups treated with the separate antibody and IL-2 are exposed to antibody for a much longer time than Leul6VhY/VkZ-1L2 due to its much longer half-life.
Furthermore, the amounts of IL-2 used for comparison were based on mass and not on IL-2 activity units. As shown in Table 2 above, free rIL-2 is approximately 3-fold more active than the equivalent molar amount of IL-2 contained in Leul6VhY/V1a-1L2 when measured with a mouse cell line expressing the high affinity IL-2R. For mouse immune cells expressing only the intermediate 11-2R, this difference is more than 10-fold in favor of free rIL-2.

Example 7. Antitumor activity of Leul6VhY, VIZ molecules in mice reconstituted with human immune cells In the SCID model reconstituted with human B cells, mice were injected i.v. with 5 x 106 CD20+ Daudi lymphoma cells on day 0 and 4.5 x human PBMC on day 5. One group (n=8) of mice received PBS only; one group received antibody only (500 mg on day 7, 9 and 11); one group received immunocytokine only (20 mg on days 11-15); and one group received the combination of antibody (500 mg on day 7, 9 and 11) and immunocytokine (20 mg on days 11-15).
All mice were checked for the presence of human antibodies in their serum by anti-human IgG ELISA on day 21 and 34. Targeting CD20 on B lymphoma cells is complicated by the fact that the antigen is expressed on normal B cells. Thus, therapy involves the targeted depletion of tumor cells in the background of a vast number of normal B cells. Since IL-2 immunocytokine dosing is likely to be limited by the toxicity of the 1L-2 component, it is unlikely that the high doses required for normal B
cell depletion by the naked antibody could be used for Leul6VhY, VKZ-1L2.
Combination treatment is a likely clinical approach in which Leul6VhY, VKZ-1L2 therapy would follow rituximab treatment to first de-bulk both CD20+ tumor cells and normal B cells. In an attempt to create a more realistic tumor model mice were injected with PBMC containing human B cells and then compared monotherapy with rituxirnab or Leul6VhYNkZ-1L2 as well as the combination in which the antibody is given as a single dose at day 7, followed by a course of therapy with Leu16VhYNkZ-IL2 beginning on day 11. A second set of mice were not implanted with human PBMC
but received the same treatment regimens. Confirmation that B cells had been implanted was obtained by measuring levels of human IgG in all mice groups. Data in Table 3 show that mice receiving human PBMC all had levels of human IgG of >500 ug/ml demonstrating efficient grafting.
Table 3. Human antibody production in SOD mice implanted with human PBMC
Day 21 Day 34 Treatment Group -B Cells + B Cells - B Cells + B Cells PBS NT >500 0 >500 DI-Leu16-1L2 (dl 1-15) NT 64.1 0.83 88.31 Rituxirnab (d7) NT 14.32 9.12 6.18 Combination NT 9.55 6.0 5.8 Human antibodies were quantitated by anti-IgG ELISA and represent mcg/ml.
Antibodies detected in the ¨ B cell group show the contribution of rituximab and DI-Leu16-1L2 (both human IgG) to the total circulating antibody .Antibody levels at day 21 show dramatic decreases in all treatment groups, including monotherapy with Leu16VhYNkZ-IL2, however the IgG level increased slightly by day 34 indicating continued production by B cells. Treatment with rituximab or the combination, on the other hand, resulted in elimination of human antibody production by day 34.
The remaining levels detected in the blood of mice in both rituximab and combination treated groups were clearly rituximab itself, since the same levels were seen in the corresponding groups that were not implanted with PBMC. Results also showed that the anti-tumor activities of all treatment groups were not significantly affected by the presence of CD20+ human B cells (Figure 15). Without wishing to be bound by theory, this may be due in part to the ability of Leul6VhYNkZ-1L2 alone to eliminate the majority of implanted B cells, as well as tumor cells. The level of activity of Leul6VhYNkZ-1L2 in this model was markedly reduced compared to earlier experiments due to the delay in initiating treatment (day 11 vs. day 7) however, it should be noted that only a single course of treatment was used to compare different molecules and additional cycles of treatment would be used clinically. We also found that the combination of a single dose of rituximab, followed by a single course of Leul6VhY/VkZ-1L2, had at least additive anti-tumor activity with the majority of mice remaining disease free at the end of the experiment (day 120).
Example 8. General methods for removing T cell epitopes from anti-CD20 antibodies Haisma et al. (Blood 92:184 (1998)) have described another anti-CD20 antibody, termed 1H4, whose V regions are about 95% identical to the V regions of Leu16. Figure 16 shows an alignment of the heavy and light (kappa) V regions of 2B8, Leu16, their epitope-depleted derivatives according to the invention, and 1H4. From the finding that the antibodies 2B8, Leu16, and 1H4 are so similar in sequence, it is an inference of the invention that all of these antibodies recognize the same epitope in CD20.
Without wishing to be bound by theory, it is an insight of the invention that CD20 is a highly conserved protein between mouse and humans, and that there is a sequence CEPANPSEKNSPSTQYC
(SEQ ID NO: 38) in the extracellular domain of CD2O that corresponds to the sequence CEPSNSSEKNSPSTQYC (SEQ ID NO: 29) in mouse CD20. In human CD20, the sequence NPS is not N-glycosylated, while the corresponding sequence NSS in mouse CD20 is N-glycosylated. According to this theory of the invention, the lack of N-glycosylation in the human CD20 reveals an antibody epitope that is absent in mouse CD20, so that when mice are immunized with human CD20, this epitope will not be recognized as self by the mouse immune system. Thus, according to the invention, the starting V regions of a monoclonal antibody directed against CD20 are at least 80%
5 identical in sequence, and more often at least 90% identical in sequence, to Leul6 or 2B8 V
regions. The antibody 1H4 is one such example. According to the invention, T
cell epitopes in an anti-CD20 antibody, for example 1H4, are removed by first aligning the heavy and light chain V regions with Leul6, 2B8, and the corresponding epitope-depleted variants shown in Figure 16, and secondly by introducing mutations in the V
regions of the 10 CD20 antibody, such as 1114, that correspond to the mutations introduced in epitope-depleted versions of Leul6 and/or 2B8. The alignments may be performed by manual alignment or using well-known alignment programs such as BLAST. For example, using the alignments in Figure 16, it is apparent that an anti-CD20 Vh domain with one or more of the following amino acid segments is generated: SGAELKKPGAS, VSC1CASGYT, 15 LEWTGAIY, YNQKFKGKT, FKGKTTLTA, YMELSSLRS, SSLRSEDTAV, and DWGTGTTVT i.e., SEQ ID NO: 15-22, respectively. Similarly, using the alignments in Figure 16, it is apparent that an anti-CD20 VL domain with one or more of the following amino acid segments is generated: IITASPGEKV, CRASTSASY, QQKPTSSP, ,LASGVPSRF, FSGSGSGTT, and YSMTISSLE, i.e., SEQ ID NO: 23-28, respectively. In 20 particular, a variant of the antibody 1114 with one or more of the preceding amino acid segments of this paragraph is generated.The utility of the newly introduced mutations may be further verified by checking that T cell epitopes are indeed removed, by using standard methods for detection of T cell epitope activity. For example, methods such as, those of Carr etal. (US Patent Publication 20030153043 and International Patent Publication WO 02/069232) may 25 be used. Alternatively, peptides corresponding to potential T cell epitopes may be added to immune cells, preferably human immune cells, and tested for the ability to sumuiate cat proliferation according to standard techniques. The corresponding V region-containing proteins are configured as whole antibodies, antibody fusion proteins, Fabs, Fab fusion proteins, single-chain Fv proteins, or other standard configurations of antibody V regions.
30 The corresponding V region-containing proteins are then produced according to standard protein expression methods, such as are described in the Examples above.

SEQUENCE LISTING
<110> Merck Patent GmbH
<120> CD2O-Binding Polypeptide Compositions <130> P03/157-bz <140> PCT/EP2004/009033 <141> 2004-08-16 <160> 43 <170> PatentIn version 3.3 <210> 1 <211> 121 <212> PRT
<213> Artificial <220>
<223> Heavy chain V region of the 2B8 antibody <400> 1 Gin Val Gin Leu Gin Gin Pro Gly Ala Glu Leu Val Lys Ala Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gin Thr Pro Gly Arg Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Thr Tyr Phe Asn Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala <210> 2 <211> 121 <212> PRT
<213> Artificial <220>
<223> Heavy chain V region VhC

<400> 2 Gin Val Gin Leu Gin Gin Pro Gly Ala Glu Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gin Thr Pro Gly Arg Gly Leu Glu Trp Thr Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala <210> 3 <211> 121 <212> PRT
<213> Artificial <220>
<223> Heavy chain V region VhD
<400> 3 Gin Val Gin Leu Gin Gin Pro Gly Ala Glu Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gin Thr Pro Gly Arg Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala <210> 4 <211> 106 <212> PRT
<213> Artificial <220>
<223> Light chain V region of the 2B8 antibody <400> 4 Gin Ile Val Leu Ser Gin Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile His Trp Phe Gin Gin Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys <210> 5 <211> 106 <212> PRT
<213> Artificial <220>
<223> Light chain V region VkA
<400> 5 Gin Ile Val Leu Ser Gin Ser Pro Ala Ile Ile Thr Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Thr Ser Ala Ser Tyr Ile His Trp Phe Gin Gin Lys Pro Thr Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Thr Tyr Ser Met Thr Ile Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys ' <210> 6 <211> 106 <212> PRT
<213> Artificial <220>
<223> Light chain V region VkB
<400> 6 Gin Ile Val Leu Ser Gin Ser Pro Ala Ile Ile Thr Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Thr Ser Val Ser Tyr Ile His Trp Phe Gin Gln Lys Pro Thr Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Thr Tyr Ser Met Thr Ile Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys <210> 7 <211> 106 <212> PRT
<213> Artificial <220>
<223> Light chain V region VkC
<400> 7 Gin Ile Val Leu Ser Gin Ser Pro Ala Ile Ile Thr Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Thr Ser Val Ser Tyr Ile His Trp Phe Gin Gin Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Thr Tyr Ser Met Thr Ile Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys <210> 8 <211> 106 <212> PRT
<213> Artificial <220>
<223> Light chain V region VkD
<400> 8 Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Ile Thr Ala Ser Pro Gly Glu Lys Val Thr Net Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Thr Tyr Ser Met Thr Ile Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys <210> 9 <211> 122 <212> PRT
<213> Artificial <220>
<223> Heavy chain V region of the Leu-16 antibody <400> 9 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Net His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Asp Tyr Tyr Cys Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp Phe Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser <210> 10 <211> 122 <212> PRT
<213> Artificial <220>
<223> Heavy chain V region VhY
<400> 10 Glu Val Gin Leu Gin Gin Ser Gly Ala Glu Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gin Thr Pro Gly Gin Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp Phe Phe Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser <210> 11 <211> 106 <212> PRT
<213> Artificial <220>
<223> Light chain V region of the Leu-16 antibody <400> 11 Asp Ile Val Leu Thr Gin Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Asp Trp Tyr Gin Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys <210> 12 <211> 106 <212> PRT
<213> Artificial <220>
<223> Light chain V region VLZ
<400> 12 Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Ile Thr Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Asp Trp Tyr Gln Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Thr Tyr Ser Met Thr Ile Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys <210> 13 <211> 123 <212> PRT
<213> Artificial <220>
<223> Heavy chain V region of the 1H4 antibody <400> 13 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Val His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Phe Asn Gin Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Val Trp Phe Phe Asp Val Trp Gly Ala Gly Thr Thr Gly Thr Gly Ser Ser <210> 14 <211> 106 <212> PRT
<213> Artificial <220>
<223> Light chain V region of the 1H4 antibody <400> 14 Gin Ile Val Leu Ser Gin Ser Pro Thr Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asp Trp Tyr Gin Gin Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp Ile Ser Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys <210> 15 <211> 11 <212> PRT
<213> Artificial <220>
<223> A first heavy chain V region segment of an anti-CD20 V region pair <400> 15 Ser Gly Ala Glu Leu Lys Lys Pro Gly Ala Ser <210> 16 <211> 9 <212> PRT
<213> Artificial <220>
<223> A second heavy chain V region segment of an anti-CD20 V region pair <400> 16 Val Ser Cys Lys Ala Ser Gly Tyr Thr <210> 17 <211> 8 <212> PRT
<213> Artificial <220>
<223> A third heavy chain V region segment of an anti-CD20 V region pair <400> 17 Leu Glu Trp Thr Gly Ala Ile Tyr <210> 18 <211> 9 <212> PRT
<213> Artificial <220>
<223> A fourth heavy chain V region segment of an anti-CD20 V region pair <400> 18 Tyr Asn Gin Lys Phe Lys Gly Lys Thr <210> 19 <211> 9 <212> PRT
<213> Artificial <220>
<223> A fifth heavy chain V region segment of an anti-CD20 V region pair <400> 19 Phe Lys Gly Lys Thr Thr Leu Thr Ala <210> 20 <211> 9 <212> PRT
<213> Artificial <220>
<223> A sixth heavy chain V region segment of an anti-CD20 V region pair <400> 20 Tyr Met Glu Leu Ser Ser Leu Arg Ser <210> 21 <211> 10 <212> PRT
<213> Artificial <220>
<223> A seventh heavy chain V region segment of an anti-CD20 V region pair <400> 21 Ser Ser Leu Arg Ser Glu Asp Thr Ala Val <210> 22 <211> 9 <212> PRT
<213> Artificial <220>
<223> A first light chain V region segment of an anti-CD20 V region pair <400> 22 Asp Trp Gly Thr Gly Thr Thr Val Thr <210> 23 <211> 10 <212> PRT
<213> Artificial <220>
<223> A second light chain V region segment of an anti-CD20 V region pair <400> 23 Ile Ile Thr Ala Ser Pro Gly Glu Lys Val <210> 24 <211> 9 <212> PRT
<213> Artificial <220>
<223> A third light chain V region segment of an anti-CD20 V region pair <400> 24 Cys Arg Ala Ser Thr Ser Ala Ser Tyr <210> 25 <211> 8 <212> PRT
<213> Artificial <220>
<223> A fourth light chain V region segment of an anti-CD20 V region pair <400> 25 Gin Gin Lys Pro Thr Ser Ser Pro <210> 26 <211> 9 <212> PRT
<213> Artificial <220>
<223> A fifth light chain V region segment of an anti-CD20 V region pair <400> 26 Leu Ala Ser Gly Val Pro Ser Arg Phe <210> 27 <211> 9 <212> PRT
<213> Artificial <220>
<223> A sixth light chain V region segment of an anti-CD20 V region pair <400> 27 Phe Ser Gly Ser Gly Ser Gly Thr Thr <210> 28 <211> 9 <212> PRT
<213> Artificial <220>
<223> A seventh light chain V region segment of an anti-CD20 V region pair <400> 28 Tyr Ser Met Thr Ile Ser Ser Leu Glu <210> 29 <211> 17 <212> PRT
<213> Artificial <220>
<223> Immunogenic Epitope Peptides of CD20 <400> 29 Cys Glu Pro Ala Asn Pro Ser Glu Lys Asn Ser Pro Ser Thr Gin Tyr Cys <210> 30 <211> 9 <212> PRT
<213> Artificial <220>
<223> Immunogenic Epitope Peptides of CD20 <400> 30 Met Ser Cys Lys Ala Ser Gly Tyr Thr <210> 31 <211> 9 <212> PRT
<213> Artificial <220>
<223> Immunogenic Epitope Peptides of CD20 <400> 31 Tyr Asn Gin Lys Phe Lys Gly Lys Ala <210> 32 <211> 9 <212> PRT
<213> Artificial <220>
<223> Immunogenic Epitope Peptides of CD20 <400> 32 Phe Lys Gly Lys Ala Thr Leu Thr Ala <210> 33 <211> 9 <212> PRT
<213> Artificial <220>
<223> Immunogenic Epitope Peptides of CD20 <400> 33 Tyr Met Gin Leu Ser Ser Leu Arg Ser <210> 34 <211> 10 <212> PRT
<213> Artificial <220>
<223> Immunogenic Epitope Peptides of CD20 <400> 34 Ile Leu Ser Ala Ser Pro Gly Glu Lys Val <210> 35 <211> 10 <212> PRT
<213> Artificial <220>
<223> Immunogenic Epitope Peptides of CD20 <400> 35 Leu Ala Ser Gly Val Pro Val Ala Arg Phe <210> 36 <211> 9 <212> PRT
<213> Artificial <220>
<223> Immunogenic Epitope Peptides of CD20 <400> 36 Phe Ser Gly Ser Gly Ser Gly Thr Ser <210> 37 <211> 9 <212> PRT
<213> Artificial <220>
<223> Immunogenic Epitope Peptides of CD20 <400> 37 Tyr Ser Leu Thr Ile Ser Arg Val Glu <210> 38 <211> 17 <212> PRT
<213> Artificial <220>
<223> Immunogenic Epitope Peptides of CD20 <400> 38 Cys Glu Pro Ser Asn Ser Ser Glu Lys Asn Ser Pro Ser Thr Gin Tyr Cys <210> 39 <211> 992 <212> DNA
<213> Artificial <220>
<223> DNA sequence encoding the mature light chain of Leul6 comprising Leul6VK
<400> 39 gacatcgttc tgacccagtc tccagcaatc ttgtctgcat ctccagggga gaaggtcacc 60 atgacctgca gagccagctc aagtgtaaat tacatggact ggtaccagaa gaagccaggc 120 tcctccccca aaccttggat ttatgccaca tccaacctgg cttctggagt ccctgctcgc 180 ttctctggca gtgggtctgg gacctcttac tctctcacaa tcagcagagt cgaggctgaa 240 gatgctgcca cttattactg ccagcagtgg agcttcaacc cacccacgtt cggtggtggg 300 accaagctgg agatcaaacg taagtggatc ccgcaattct aaactctgag ggggtcggat 360 gacgtggcca ttctttgcct aaagcattga gtttactgca aggtcagaaa agcatgcaaa 420 gccctcagaa tggctgcaaa gagctccaac aaaacaattt agaactttat taaggaatag 480 ggggaagcta ggaagaaact caaaacatca agattttaaa tacgcttctt ggtctccttg 540 ctataattat ctgggataag catgctgttt tctgtctgtc cctaacatgc cctgtgatta 600 tccgcaaaca acacacccaa gggcagaact ttgttactta aacaccatcc tgtttgcttc 660 tttcctcagg aactgtggct gcaccatctg tcttcatctt cccgccatct gatgagcagt 720 tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc agagaggcca 780 aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag agtgtcacag 840 agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg agcaaagcag 900 actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg agctcgcccg 960 tcacaaagag cttcaacagg ggagagtgtt ag 992 <210> 40 <211> 992 <212> DNA
<213> Artificial <220>
<223> DNA sequence encoding an epitope-depleted variant of the mature light chain of Leul6 comprising Leu16VKZ
<400> 40 gacattgttc tcacccagtc tccagcaatc atcacagcat ctccagggga gaaggtcaca 60 atgacttgca gggccagctc aagtgtaaac tacatggact ggtaccagaa gaagccaggg 120 tcctccccca aaccctggat ttatgccaca tccaacctgg cttctggagt cccttctcgc 180 ttcagtggca gtgggtctgg gactacttac tctatgacca tcagcagcct cgaggctgaa 240 gatgctgcca cttattactg ccagcagtgg agcttcaacc cacccacgtt cggagggggg 300 accaagctgg aaatcaaacg taagtggatc ccgcaattct aaactctgag ggggtcggat 360 gacgtggcca ttctttgcct aaagcattga gtttactgca aggtcagaaa agcatgcaaa 420 gccctcagaa tggctgcaaa gagctccaac aaaacaattt agaactttat taaggaatag 480 ggggaagcta ggaagaaact caaaacatca agattttaaa tacgcttctt ggtctccttg 540 ctataattat ctgggataag catgctgttt tctgtctgtc cctaacatgc cctgtgatta 600 tccgcaaaca acacacccaa gggcagaact ttgttactta aacaccatcc tgtttgcttc 660 tttcctcagg aactgtggct gcaccatctg tcttcatctt cccgccatct gatgagcagt 720 tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc agagaggcca 780 aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag agtgtcacag 840 agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg agcaaagcag 900 actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg agctcgcccg 960 tcacaaagag cttcaacagg ggagagtgtt ag 992 <210> 41 <211> 2184 <212> DNA
<213> Artificial <220>
<223> DNA sequence encoding the mature heavy chain of Leul6 comprising Leul6VH
<400> 41 gaggtccagc tccagcagtc tggagctgag ctggtgaagc ctggggcttc agtgaagatg 60 tcctgcaagg cttctggata cacattcact agttataata tgcactgggt aaagcagaca 120 cctggacagg gcctggaatg gattggagct atttatccag gaaatggtga tacttcctac 180 0861 SPE,BlEoDED TeopEo8poo oTeqpqq366 peppq66qop 6qoop6qop6 poq55poopp OZ61 Ecepoop&Teb pE5PEE6Doo qpooppoBqD op.eop.46.46.6 poPopEE5P6 pooDBED666 0981 Paegpopq61 ogooPupopq Eqp5oop616 uBuEgooDE.q. oqopoppoo6 Bogo6Eop66 0081 P6PDPEEc4PD eopE5Ece5o8 4.6655q5Dpo p666.466pET. 006PPUDDqD
TeDDEUEUECe OtGT 5p;Poopoo6 PopogoopEce upoppoowq 55p 55 oug5pE6pPo 66Tep671DE6 0891 goubaeopup EqoagEoppo googEoEcepq 65g5gEoppl BDUDSPOPPO pq6pD6p66P
OZ91 665pEop6p8 ppuE,ppoobq upTepEqE.Bp 6.6q6DE6ae6 6.46opq864o PupqqaePoq 09S1 EBP6qopoy5 upEopoo6p5 qEopE6q66q 58q6p6Tepe oq65p6qoop oPEEopowq 00ST P54-epqopop DUEE.PPOODP PPPODOODOq qoqopqqp4E. uogEopu66.6 666qopqopp Otti 6qopPo6epq opqqpqpqpo owppopq6o ppu6qp6;66 Eop6uppop5 .6-epu666Poo 08E1 TepaqopEceq 6u6pqoppEcq 66pop666p6 BPPOqD6P00 qop36ogoo6 Ecepop6pDo6 ()ZEE P-eq6Ecepoo6 qBooppooBq POPOPOWPP ppouBqEqqo TePpooDEPE, Pa6wwwq 09Z1 qoqPPoopqo ppq6uppqqp Buppowoqo qpqqpppopE. EowEceogop pqoppogow 0OZT PPPODBEcePP 0000E0006P pqoppEcloop D6qopop66-E. 66633Tel-ea pErpEcepoo6q OtTT poPE.ppw66 66666 666-epupupp 36qopo6Ecep Doppqoppo6 466-egobEcep 0801 Po66-ep666q o366poppo qqqqqobbqo qqpq566pEce 655uoqp6Te ogouppopEo OZOT opEcwqop65 66 DD
oqopEcqpq6o oDDE6P365-e ppEcep666-ep Dq6pooDT6p 096 p5qpqp6Eop pqppEou.56.3 opEcqopqp6o SpowEE.upp 6ppEEE.qo q645E6a666 006 p666poppEce op6Be5p6.36 Eqq6pEce6uu OPEZqBE,PUD DPOPPDBUDD DEIPPOPOqUP
Ot8 6q6oupo6qo quaegoop6u poppoBE5T4 pEcepEcepogo po6gEopp6q 66q5p6po6u 08G oqopoqouqp qopEBuogoo .46-eopqopq6 qp6Boopqqo opoPpEcq6DE 6o6upop6qo On opEo66-e3go ppE.E.gEogaq 6EopEq.5.6op ppEopooqqo uqopEZeupq 66qopEqp66 099 610336605P 3pp66666.43 WDPD6P6PP ODWO;DODP DE,Eqppoopq wq6BoTeop 009 3556ppo32o 3q336p3644 oqpqoppoop o66gEopagE. 6060DPOPOD ogaqogoEcep OtS op666qoppE. 06qpqD3E55 EceopoPuBuu qq6eop5636 o33ep6q35 3p66qoppu5 08t p3336u6Te3 DOBTeUDDDP op35.166p33 5po3E356q6 Ece35BeEq.56 ppqp66565p OZt E55PDE666q qqa6Eqqopp 6wo66-eDD5 Eceo5E65qpq qqqp6-2-eq6u pq66poqwq 09E 3gEo3Poq66 oppop.666up 6o5655qpqE. Te6oggoqq5 Eqouqp6p46 PqE,Eopqoeq 00E opeq.E.PE5p6 p6q6qopqqu qopBqobwq a5 5t oubqppEcepE. poqp5ua6Te OtZ DP4006PDPD E.P00qDDT2P p3u5p36q3p EqopaeDDE6 ppoBEZ-eupq q&epaeoqup tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 2040 gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 2100 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 2160 ctctccctgt ccccgggtaa atga 2184 <210> 42 <211> 2179 <212> DNA
<213> Artificial <220>
<223> DNA sequence encoding an epitope depleted-variant of the mature heavy chain of Leul6 comprising Leu16VHY
<400> 42 gaggtacaac tgcagcagtc tggggctgag ctgaagaagc ctggggcctc agtgaaggtg 60 tcctgcaagg cttctggcta cacatttacc agttacaata tgcactgggt aaaacagaca 120 cctggtcagg gcctggaatg gattggagct atttatcccg gaaatggtga tacttcctac 180 aatcagaagt tcaaaggcaa gacaacattg actgcagaca aatcctccag cacagcctac 240 atggaactca gcagcctgag atctgaggac actgcggtct attactgtgc aagatcgaat 300 tactacggca gcagctactg gttcttcgat gtctggggca ccgggaccac ggtcaccgtc 360 tcttcaggta agctttctgg ggcaggccag gcctgacctt ggctttgggg cagggagggg 420 gctaaggtga ggcaggtggc gccagccagg tgcacaccca atgcccatga gcccagacac 480 tggacgctga acctcgcgga cagttaagaa cccaggggcc tctgcgccct gggcccagct 540 ctgtcccaca ccgcggtcac atggcaccac ctctcttgca gcctccacca agggcccatc 600 ggtcttcccc ctggcaccct cctccaagag cacctctggg ggcacagcgg ccctgggctg 660 cctggtcaag gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac 720 cagcggcgtg cacaccttcc cggctgtcct acagtcctca ggactctact ccctcagcag 780 cgtggtgacc gtgccctcca gcagcttggg cacccagacc tacatctgca acgtgaatca 840 caagcccagc aacaccaagg tggacaagag agttggtgag aggccagcac agggagggag 900 ggtgtctgct ggaagccagg ctcagcgctc ctgcctggac gcatcccggc tatgcagtcc 960 cagtccaggg cagcaaggca ggccccgtct gcctcttcac ccggaggcct ctgcccgccc 1020 cactcatgct cagggagagg gtcttctggc tttttcccca ggctctgggc aggcacaggc 1080 taggtgcccc taacccaggc cctgcacaca aaggggcagg tgctgggctc agacctgcca 1140 agagccatat ccgggaggac cctgcccctg acctaagccc accccaaagg ccaaactctc 1200 cactccctca gctcggacac cttctctcct cccagattcc agtaactccc aatcttctct 1260 ctgcagagcc caaatcttgt gacaaaactc acacatgccc accgtgccca ggtaagccag 1320 cccaggcctc gccctccagc tcaaggcggg acaggtgccc tagagtagcc tgcatccagg 1380 gacaggcccc agccgggtgc tgacacgtcc acctccatct cttcctcagc acctgaactc 1440 ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 1500 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 1560 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 1620 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 1680 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1740 accatctcca aagccaaagg tgggacccgt ggggtgcgag ggccacatgg acagaggccg 1800 gctcggccca ccctctgccc tgagagtgac cgctgtacca acctctgtcc ctacagggca 1860 gccccgagaa ccacaggtgt acaccctgcc cccatcacgg gaggagatga ccaagaacca 1920 ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga 1980 gagcaatggg cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg 2040 ctccttcttc ctctatagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt 2100 cttctcatgc tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc 2160 cctgtccccg ggtaaatga 2179 <210> 43 <211> 2578 <212> DNA
<213> Artificial <220>
<223> DNA sequence encoding a fusion protein of an epitope-depleted variant of the mature heavy chain of Leul6 comprising Leu16VHY
and the cytokine IL2 <400> 43 gaggtacaac tgcagcagtc tggggctgag ctgaagaagc ctggggcctc agtgaaggtg 60 tcctgcaagg cttctggcta cacatttacc agttacaata tgcactgggt aaaacagaca 120 cctggtcagg gcctggaatg gattggagct atttatcccg gaaatggtga tacttcctac 180 aatcagaagt tcaaaggcaa gacaacattg actgcagaca aatcctccag cacagcctac 240 atggaactca gcagcctgag atctgaggac actgcggtct attactgtgc aagatcgaat 300 tactacggca gcagctactg gttcttcgat gtctggggca ccgggaccac ggtcaccgtc 360 tcttcaggta agctttctgg ggcaggccag gcctgacctt ggctttgggg cagggagggg 420 gctaaggtga ggcaggtggc gccagccagg tgcacaccca atgcccatga gcccagacac 480 08zz PPD3D1PP6P POPqOPV0PP qweEZTE,P6 goqq-e6TeSP pogoqP56.4o 64opqogyo6 ozu P5Epqovrobq 06PDPOPPPP 6E-epop33qq EisPoqqopvp opoBvp6q5E. 600ppe6pEo 091z oppo5a6.26-E, pEcep6opovq. DPOOPPOPOS qoqp6EsSqu ofq..e6T6opq DETeoqoqqo 00-Ez 1.5aevE,666E. DE.p366q65E. p6p6pvae66 45oppoqp5e. pobvquqoqo oqqoqqopqo 0t,oz EbovEopqoP BE,qp5q6pop qp060PDDPE. PEDPqOPPOP p6v6EopEcep BEBTeppEce6 0861 p6.66q6v65q. 60063quae6 abuoppiplo qqp56uppo1 664305.433v 6q335-e3qb.6 (:)61 P33PP6PPOD P5qP5ce.6.6P5 560POTe000 oo6qopoupP 4.6;66-eoppo PP6a600006 0981 Pp656vou43 opqMoioop eopeq6q363 op6q6p6v6q 3ppEr4olopo v3p36Eolo6 008T 5oo5Ere5Pop 6Elquppop65 EcebabgBEZE. gEoppuB65q 66E-evoDEPP poololpoop otLT PPP6P60qPD DOODSPODO1 DODBPVPOPP Dowq6Ecepo 6qEcepopq5p E.E.E.PoarqPE, 0891 .61356qoP.66 spopoSqopq boopogooqb o6p3q.6.6q6.4 630"eq.63PDB

0z91 6PE5E66606 Do5P-eups5, poobTeuquo 63E6p66q6o 5Eop56q6DP q6E,goepoqq 09s1 .6-2.2o466-e51 opouBppEoe 336v5;6325 6q661.6.64.63 .6Teovoi.6.6.e.
Eqoppae6Bo oosT powq.e5qPo qoppeopbEce PODOPPPPOD oppoqqpqop qwq6Paq6o oPEZEZBEqp 0f717I Dq3pP64Dou obvpqopqw ioqeopqope poqbaeovbq p64655o36y p00056pae6 BET BEPooTeoBq Da6pq5E,Ereq Doo64.6.6pop 66606.6.evoq DEcepoqpoo6 oqoa6.6.epoo ozu 6-epoBvt,166 popoBgEopy 33p6qp3eou oq3pureppy5 qbqqp.TePpo DoBeEleobio 09Z1 qoqoqq.D.Tep opoqopeq6p poTTE,Bpoop qopwwqqo opovBEoqa6 poqopoqopo clozT ogoqp.epPoo 6.6epuppopv opoBvpqopu 6qopoo6qop 3-266E6E63o q-equopbeEfe of,TT Poo6goopEce oqp66.6qoBq 5.6.2o55.65-ep vopoppEgoo obErepooppq opopEcq5E.E.3 10801 p6.popo66p o666-qoqo5b voopoqqqqq obbgaqqoqE, 66p6v666po qp&TepqaPo ozoT oppEopoBqp qop56p65oo opoqqpipab qpq6poop55 Po66pPp5eo Eacepoq6up 096 pogaeo&Teq o65oopTeD6 op66qopEcgo oqp6pEceow 5E,vopEcep66 qp5qoq5456 006 6pE65y6E6u ovo6Poo56E, bp.6466.11.Bp 6p5uppe56q acePOOPOPP obpoopbepo 0f18 upgpsEq.Bop po6qoqpovq o3p6spoppo 566qqo6po6 ppoqopp6q6 DoP6q66.46o 08L .6Po5vpqppo goviogova, u3gool6Pop qo3q.51o663 opqqoppopo 6q6D56.7)5u3 OZL op5qopo5a6 EceoqopuBbq 5DT6g5Eou5 46.6paevEop poqqouqop6 Erepoq65wo 099 6.43666.4pop 6636popo66 655qoqoppo 5p6ppooqop qoppypaqo opppgloq66 009 oTeoppa,Bu pooppowDE, po6.4.4ogogo oppoPp66.4.2 opo.4653.6po yoppooqBqo ()VS go5poop.666 qopo6p6qpq 336665-ep3p vs5yugq6po u66pEoqoae. v6qp6ov6&q actcaccagg atgctcacat tcaagttcta catgcccaag aaggccacag agctcaaaca 2340 tctccagtgt ctagaggagg aactcaaacc tctggaggaa gtgctaaacc tcgctcagag 2400 caaaaacttc cacttaagac ctagggactt aatcagcaat atcaacgtaa tagttctgga 2460 actaaaggga tccgaaacaa cattcatgtg tgaatatgct gatgagacag caaccattgt 2520 agaattccta aacagatgga ttaccttttg tcaaagcatc atctcaacac taacttga 2578

Claims (18)

CLAIMS:
1. An anti-CD20 antibody molecule fused to IL-2 comprising:
(i) a modified heavy chain variable region polypeptide having the amino acid residue sequence of SEQ ID NO: 9, but which includes an amino acid residue substitution in said sequence selected from the group consisting of V12K, M20V, A68T, Q82E, 1-87R, S91T, D93V, and A114T, (ii) a modified light chain variable region polypeptide having the amino acid residue sequence of SEQ ID NO: 11, but which includes an amino acid residue substitution in said sequence selected from the group consisting of L11I, S12T, A59S, 569T, L72M, R76S, and V77L, and (iii) a human IgG heavy chain constant region and a human light chain constant region.
2. The anti-CD20 antibody fusion protein of claim 1, wherein the IgG
constant region is a human IgG1 constant region.
3. The anti-CD20 antibody fusion protein of claim 1 or 2, wherein the human light chain constant region is a human kappa light chain constant region.
4. The anti-CD20 antibody fusion protein according to any one of the claims 1-3 comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10.
5. The anti-CD20 antibody fusion protein according to any one of the claims 1-3 comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12.
6. The anti-CD20 antibody fusion protein according to any one of the claims 1-3 comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12.
7. The anti-CD20 antibody fusion protein according to any one of the claims 1-6, wherein the C-terminus of the antibody molecule is fused to the N-terminus of IL-2.
8. The anti-CD20 antibody fusion protein according to claim 1 encoded by the DNA sequences of SEQ ID NO: 40 and SEQ ID NO: 43.
9. A pharmaceutical composition comprising the anti-CD20 antibody fusion protein according to any one of the claims 1-8, together with a pharmaceutically acceptable carrier, excipient or diluent.
10. The pharmaceutical composition of claim 9 further comprising an additional pharmacologically effective drug.
11. A DNA molecule coding for the anti-CD20 antibody fusion protein according to any one of the claims 1-8.
12. Use of the anti-CD20 antibody fusion protein of any one of the claims 1-8 or the pharmaceutical composition of claim 9 or 10, for the manufacture of a medicament for the treatment of CD20 expressing cancers.
13. Use of claim 12, wherein said cancer is B-cell lymphoma.
14. Use of the anti-CD20 antibody fusion protein of any one of the claims 1-8 for the treatment of CD20 expressing cancers.
15. Use of claim 14, wherein said cancer is B-cell lymphoma.
16. The pharmaceutical composition of claim 9 or 10 for use in the treatment of CD20 expressing cancers.
17. The pharmaceutical composition of claim 16, wherein said cancer is B-cell lymphoma.
18. The pharmaceutical composition of claim 10 comprising the anti-CD20 antibody fusion protein of claim 8 and monoclonal antibody rituximab.
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Families Citing this family (67)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1802388B (en) * 2003-05-09 2011-01-05 杜克大学 CD20-specific antibodies and methods of employing same
US8147832B2 (en) * 2003-08-14 2012-04-03 Merck Patent Gmbh CD20-binding polypeptide compositions and methods
WO2005120437A2 (en) * 2004-06-04 2005-12-22 Genentech, Inc. Method for treating lupus
AU2005249566B2 (en) * 2004-06-04 2010-11-11 Genentech, Inc. Method for treating multiple sclerosis
KR20070036187A (en) * 2004-07-22 2007-04-02 제넨테크, 인크. Method of treating sjogren's syndrome
KR100864549B1 (en) 2004-08-04 2008-10-20 어플라이드 몰리큘라 에볼류션, 인코포레이티드 Variant fc regions
WO2006041680A2 (en) * 2004-10-05 2006-04-20 Genentech, Inc. Method for treating vasculitis
AR053579A1 (en) * 2005-04-15 2007-05-09 Genentech Inc TREATMENT OF INTESTINAL INFLAMMATORY DISEASE (IBD)
CN101223448B (en) * 2005-05-20 2012-01-18 健泰科生物技术公司 Pretreatment of a biological sample from an autoimmune disease subject
MY149159A (en) 2005-11-15 2013-07-31 Hoffmann La Roche Method for treating joint damage
DK2270050T3 (en) 2005-12-30 2013-08-12 Merck Patent Gmbh Anti-CD19 antibodies with reduced immunogenicity
US9382327B2 (en) 2006-10-10 2016-07-05 Vaccinex, Inc. Anti-CD20 antibodies and methods of use
RU2474585C2 (en) 2007-01-22 2013-02-10 Дженентек, Инк. Precipitating and purifying proteins with polyelectrolytes
AU2008277907B2 (en) * 2007-07-17 2013-08-22 Merck Patent Gmbh Engineered anti-alpha V- integrin hybrid antibodies
JP2011500715A (en) 2007-10-16 2011-01-06 ザイモジェネティクス, インコーポレイテッド Combination of BLyS inhibitor and anti-CD20 agent for the treatment of autoimmune disease
CA2703279C (en) 2007-10-30 2014-04-22 Genentech, Inc. Antibody purification by cation exchange chromatography
AU2008342956A1 (en) * 2007-12-21 2009-07-09 Genentech, Inc. Therapy of rituximab-refractory rheumatoid arthritis patients
WO2009134738A1 (en) * 2008-04-29 2009-11-05 Genentech, Inc. Responses to immunizations in rheumatoid arthritis patients treated with a cd20 antibody
KR101843915B1 (en) 2008-08-14 2018-04-02 제넨테크, 인크. Methods for removing a contaminant using indigenous protein displacement ion exchange membrane chromatography
EP2684570A1 (en) 2008-09-10 2014-01-15 F. Hoffmann-La Roche AG Compositions and methods for the prevention of oxidative degradation of proteins
AR073295A1 (en) 2008-09-16 2010-10-28 Genentech Inc METHODS TO TREAT PROGRESSIVE MULTIPLE SCLEROSIS. MANUFACTURING ARTICLE.
WO2010075249A2 (en) 2008-12-22 2010-07-01 Genentech, Inc. A method for treating rheumatoid arthritis with b-cell antagonists
AR080154A1 (en) 2010-02-10 2012-03-14 Immunogen Inc CD20 ANTIBODIES AND ITS USE
SI2550018T1 (en) 2010-03-22 2019-05-31 F. Hoffmann-La Roche Ag Compositions and methods useful for stabilizing protein-containing formulations
CA2794864A1 (en) 2010-05-03 2011-11-10 Genentech, Inc. Compositions and methods useful for reducing the viscosity of protein-containing formulations
EP2585045B1 (en) 2010-06-24 2019-08-21 F.Hoffmann-La Roche Ag Compositions and methods containing alkylgycosides for stabilizing protein-containing formulations
WO2012018771A1 (en) 2010-08-03 2012-02-09 Genentech, Inc. Chronic lymphocytic leukemia (cll) biomarkers
KR20130086628A (en) 2010-11-08 2013-08-02 제넨테크, 인크. Subcutaneously administered anti-il-6 receptor antibody
EP3252076B1 (en) 2011-01-14 2019-09-04 The Regents Of The University Of California Diagnostic use of antibodies against ror-1 protein
ES2683347T3 (en) 2011-02-11 2018-09-26 Merck Patent Gmbh Anti-integrin alpha-v antibody for the treatment of prostate cancer
CN108892706A (en) 2011-12-22 2018-11-27 弗·哈夫曼-拉罗切有限公司 The method for improving downstream protein purification step using Ion exchange membrane chromatography
US9777067B2 (en) 2012-09-27 2017-10-03 Massachusetts Institute Of Technology HER2- and VEGF-A-binding proteins with enhanced stability
KR102110127B1 (en) 2013-03-12 2020-05-14 몰레큘러 템플레이츠, 인코퍼레이션. Cd20-binding immunotoxins for inducing cellular internalization and methods using same
CN105358177B (en) 2013-04-17 2018-11-23 西格诺药品有限公司 Conjoint therapy comprising TOR kinase inhibitor and IMID compound is used for treating cancer
WO2014172436A1 (en) 2013-04-17 2014-10-23 Signal Pharmaceuticals, Llc Combination therapy comprising a tor kinase inhibitor and a 5-substituted quinazolinone compound for treating cancer
US9358232B2 (en) 2013-04-17 2016-06-07 Signal Pharmaceuticals, Llc Methods for treating cancer using TOR kinase inhibitor combination therapy
JP2016521280A (en) 2013-05-03 2016-07-21 セルジーン コーポレイション How to treat cancer with combination therapy
IL285403B2 (en) 2014-01-27 2023-10-01 Molecular Templates Inc Mhc class i epitope delivering polypeptides
GB201403775D0 (en) 2014-03-04 2014-04-16 Kymab Ltd Antibodies, uses & methods
US11142584B2 (en) * 2014-03-11 2021-10-12 Molecular Templates, Inc. CD20-binding proteins comprising Shiga toxin A subunit effector regions for inducing cellular internalization and methods using same
ES2887299T3 (en) 2014-04-03 2021-12-22 Igm Biosciences Inc Modified J string
AU2015274647C1 (en) 2014-06-11 2020-01-30 Molecular Templates, Inc. Protease-cleavage resistant, Shiga toxin a subunit effector polypeptides and cell-targeted molecules comprising the same
KR101651658B1 (en) 2014-12-30 2016-08-29 (주)제이디산업 Automatic fire extinguisher and manufacturing method thereof
KR20170110601A (en) * 2015-02-05 2017-10-11 몰레큘러 템플레이츠, 인코퍼레이션. Multivalent CD20 binding molecules comprising a &lt; RTI ID = 0.0 &gt; cigarotoxin A &lt; / RTI &gt;
KR101664867B1 (en) 2015-02-17 2016-10-12 (주)제이디산업 Fire extinguisher using drone
CN107532188B (en) 2015-03-04 2022-05-10 Igm生物科学股份有限公司 CD20 binding molecules and uses thereof
EP3303373B1 (en) 2015-05-30 2020-04-08 Molecular Templates, Inc. De-immunized, shiga toxin a subunit scaffolds and cell-targeting molecules comprising the same
CN108463472A (en) 2015-09-30 2018-08-28 Igm生物科学有限公司 The binding molecule of J- chains with modification
CA2999284C (en) 2015-09-30 2023-06-13 Igm Biosciences A/S Binding molecules with modified j-chain
US10871490B2 (en) * 2016-02-29 2020-12-22 Multenyi Biotec, gmbH Assay for detection of chimeric antigen receptor T cells
US9567399B1 (en) 2016-06-20 2017-02-14 Kymab Limited Antibodies and immunocytokines
WO2018083248A1 (en) 2016-11-03 2018-05-11 Kymab Limited Antibodies, combinations comprising antibodies, biomarkers, uses & methods
CN114773439A (en) 2016-12-07 2022-07-22 分子模板公司 Shiga toxin A subunit effector polypeptides, shiga toxin effector scaffolds and cell targeting molecules for site-specific conjugation
US10610104B2 (en) 2016-12-07 2020-04-07 Progenity, Inc. Gastrointestinal tract detection methods, devices and systems
IL267990B1 (en) 2017-01-25 2023-12-01 Molecular Templates Inc Cell-targeting molecules comprising de-immunized, shiga toxin a subunit effectors and cd8 t-cell epitopes
WO2018183929A1 (en) 2017-03-30 2018-10-04 Progenity Inc. Treatment of a disease of the gastrointestinal tract with an immune modulatory agent released using an ingestible device
CA3097178A1 (en) 2018-04-17 2019-10-24 Molecular Templates, Inc. Her2-targeting molecules comprising de-immunized, shiga toxin a subunit scaffolds
US20230009902A1 (en) 2018-06-20 2023-01-12 Progenity, Inc. Treatment of a disease or condition in a tissue orginating from the endoderm
US20230041197A1 (en) 2018-06-20 2023-02-09 Progenity, Inc. Treatment of a disease of the gastrointestinal tract with an immunomodulator
CN113348011B (en) 2018-11-19 2023-04-18 比奥拉治疗股份有限公司 Method and apparatus for treating disease with biotherapeutic agents
EP3946457A1 (en) 2019-04-01 2022-02-09 Genentech, Inc. Compositions and methods for stabilizing protein-containing formulations
EP4309722A2 (en) 2019-12-13 2024-01-24 Biora Therapeutics, Inc. Ingestible device for delivery of therapeutic agent to the gastrointestinal tract
CN115315436A (en) 2020-01-10 2022-11-08 明峰治疗股份公司 Modified IL-2 polypeptides and uses thereof
WO2022235622A2 (en) * 2021-05-04 2022-11-10 Biocove Llc Cd20 targeting fusion proteins and methods of use thereof
US20230201365A1 (en) * 2021-07-09 2023-06-29 Bright Peak Therapeutics Ag Modified cd20 antibodies and uses thereof
WO2023288267A1 (en) 2021-07-14 2023-01-19 2Seventy Bio, Inc. Engineered t cell receptors fused to binding domains from antibodies
WO2023196996A2 (en) 2022-04-08 2023-10-12 2Seventy Bio, Inc. Multipartite receptor and signaling complexes

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6010902A (en) 1988-04-04 2000-01-04 Bristol-Meyers Squibb Company Antibody heteroconjugates and bispecific antibodies for use in regulation of lymphocyte activity
US5650150A (en) 1990-11-09 1997-07-22 Gillies; Stephen D. Recombinant antibody cytokine fusion proteins
AU688743B2 (en) * 1992-11-13 1998-03-19 Biogen Idec Inc. Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US6100387A (en) 1997-02-28 2000-08-08 Genetics Institute, Inc. Chimeric polypeptides containing chemokine domains
EP1156823B1 (en) 1999-02-12 2008-10-29 The Scripps Research Institute Methods for treatment of tumors and metastases using a combination of anti-angiogenic and immuno therapies
JP4286483B2 (en) * 1999-06-09 2009-07-01 イムノメディクス, インコーポレイテッド Immunotherapy for autoimmune diseases using antibodies targeting B cells
CA2438652A1 (en) * 2001-02-19 2002-09-06 Merck Patent Gesellschaft Mit Beschraenkter Haftung Method for identification of t-cell epitopes and use for preparing molecules with reeduced immunogenicity
RU2297245C2 (en) * 2001-02-19 2007-04-20 Мерк Патент Гмбх Modified anti-egfr antibodies with reduced ammonogenicity
BR0207267A (en) * 2001-02-19 2004-02-10 Merck Patent Gmbh Artificial proteins with reduced immunogenicity
WO2003061694A1 (en) * 2001-05-10 2003-07-31 Seattle Genetics, Inc. Immunosuppression of the humoral immune response by anti-cd20 antibodies
US7321026B2 (en) * 2001-06-27 2008-01-22 Skytech Technology Limited Framework-patched immunoglobulins
CA2466592A1 (en) * 2001-11-12 2003-05-22 Koen Hellendoorn Modified anti-tnf alpha antibody
CA2469045A1 (en) * 2001-12-07 2003-06-19 Chiron Corporation Methods of therapy for non-hodgkin's lymphoma
US8147832B2 (en) * 2003-08-14 2012-04-03 Merck Patent Gmbh CD20-binding polypeptide compositions and methods

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